Methods of detection of a target nucleic acid sequence

ABSTRACT

The present invention is directed to methods of generating a signal indicative of the presence of a target nucleic acid in a sample by forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid with a thermostable nucleic acid polymerase substantially lacking 5′ to 3′ exonuclease activity and cleaving said cleavage structure with a thermostable Fen nuclease lacking 5′ to 3′ synthetic activity to generate a signal.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 11/106,237, filed Apr. 14, 2005, now U.S. Pat. No.7,482,121 which is a divisional application of U.S. application Ser. No.09/728,574, now U.S. Pat. No. 7,118,860, which was filed on Nov. 30,2000. U.S. application Ser. No. 09/728,574 is a continuation-in-part ofU.S. application Ser. No. 09/650,888, filed Aug. 30, 2000 now U.S. Pat.No. 6,548,250, which is a continuation-in-part of U.S. application Ser.No. 09/430,692, filed Oct. 29, 1999, now U.S. Pat. No. 6,528,254. Theentire teachings of the above applications are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates in general to methods of detecting or measuring atarget nucleic acid sequence.

BACKGROUND OF THE INVENTION

The fidelity of DNA replication, recombination, and repair is essentialfor maintaining genome stability, and all of these processes depend on5′→3′ exonuclease enzymes which are present in all organisms. For DNArepair, these enzymes are required for damaged fragment excision andrecombinational mismatch correction. For replication, these nucleasesare critical for the efficient processing of Okazaki fragments duringlagging strand DNA synthesis. In Escherichia coli, this latter activityis provided by DNA polymerase I (PolI); E. coli strains withinactivating mutations in the Poll 5′→3′ exonuclease domain are notviable due to an inability to process Okazaki fragments. Eukaryotic DNApolymerases, however, lack an intrinsic 5′→3′ exonuclease domain, andthis critical activity is provided by the multifunctional,structure-specific metallonuclease FEN-1 (five' exonuclease-1 or flapendonuclease-1), which also acts as an endonuclease for 5′ DNA flaps(Reviewed in Hosfield et al., 1998a, Cell, 95:135).

Methods of detecting and/or measuring a nucleic acid wherein an enzymeproduces a labeled nucleic acid fragment are known in the art.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose a method of cleaving a target DNA molecule by incubating a 5′labeled target DNA with a DNA polymerase isolated from Thermus aquaticus(Taq polymerase) and a partially complementary oligonucleotide capableof hybridizing to sequences at the desired point of cleavage. Thepartially complementary oligonucleotide directs the Taq polymerase tothe target DNA through formation of a substrate structure containing aduplex with a 3′ extension opposite the desired site of cleavage whereinthe non-complementary region of the oligonucleotide provides a 3′ armand the unannealed 5′ region of the substrate molecule provides a 5′arm. The partially complementary oligonucleotide includes a 3′nucleotide extension capable of forming a short hairpin. The release oflabeled fragment is detected following cleavage by Taq polymerase.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose the generation of mutant, thermostable DNA polymerases thathave very little or no detectable synthetic activity, and wild typethermostable nuclease activity. The mutant polymerases are said to beuseful because they lack 5′ to 3′ synthetic activity; thus syntheticactivity is an undesirable side reaction in combination with a DNAcleavage step in a detection assay.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose that wild type Taq polymerase or mutant Taq polymerases thatlack synthetic activity can release a labeled fragment by cleaving a 5′end labeled hairpin structure formed by heat denaturation followed bycooling, in the presence of a primer that binds to the 3′ arm of thehairpin structure. Further, U.S. Pat. Nos. 5,843,669, 5,719,028,5,837,450, 5,846,717 and 5,888,780 teach that the mutant Taq polymeraseslacking synthetic activity can also cleave this hairpin structure in theabsence of a primer that binds to the 3′ arm of the hairpin structure.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that cleavage of this hairpin structure in the presence ofa primer that binds to the 3′ arm of the hairpin structure by mutant Taqpolymerases lacking synthetic activity yields a single species oflabeled cleaved product, while wild type Taq polymerase producesmultiple cleavage products and converts the hairpin structure to adouble stranded form in the presence of dNTPs, due to the high level ofsynthetic activity of the wild type Taq enzyme.

The 5′ to 3′ exonuclease activity of a nucleic acid polymerase canimpair the amplification of certain nucleic acids. There is also a needin the art for a method of generating a signal using a nucleic acidcleavage reaction in the absence of a 5′ to 3′ exonuclease activity of anucleic acid polymerase.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that mutant Taq polymerases exhibiting reduced syntheticactivity, but not wild type Taq polymerase, can release a single labeledfragment by cleaving a linear nucleic acid substrate comprising a 5′ endlabeled target nucleic acid and a complementary oligonucleotide whereinthe complementary oligonucleotide hybridizes to a portion of the targetnucleic acid such that 5′ and 3′ regions of the target nucleic acid arenot annealed to the oligonucleotide and remain single stranded.

There is a need in the art for a method of generating a signal of adiscrete size that can be easily distinguished from oligonucleotidefragments that may arise from nuclease contaminants, using a nucleicacid cleavage reaction in the absence of 5′ to 3′ exonuclease activityof a nucleic acid polymerase.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose a method of cleaving a labeled nucleic acid substrate atnaturally occurring areas of secondary structure. According to thismethod, biotin labeled DNA substrates are prepared by PCR, mixed withwild type Taq polymerase or CleavaseBN (a mutant Taq polymerase withreduced synthetic activity and wild type 5′ to 3′ nuclease activity),incubated at 95° C. for 5 seconds to denature the substrate and thenquickly cooled to 65° C. to allow the DNA to assume its unique secondarystructure by allowing the formation of intra-strand hydrogen bondsbetween the complementary bases. The reaction mixture is incubated at65° C. to allow cleavage to occur and biotinylated cleavage products aredetected.

There is a need in the art for a method of generating a signal using anucleic acid cleavage reaction in the absence of a 5′ to 3′ exonucleaseactivity of a nucleic acid polymerase wherein the cleavage structure isnot required to contain areas of secondary structure.

Methods of detecting and/or measuring a nucleic acid wherein a FEN-1enzyme is used to generate a labeled nucleic acid fragment are known inthe art.

U.S. Pat. No. 5,843,669 discloses a method of detecting polymorphisms bycleavase fragment length polymorphism analysis using a thermostableFEN-1 nuclease in the presence or absence of a mutant Taq polymeraseexhibiting reduced synthetic activity. According to this method, doublestranded Hepatitis C virus (HCV) DNA fragments are labeled by using 5′end labeled primers (labeled with TMR fluorescent dye) in a PCRreaction. The TMR labeled PCR products are denatured by heating to 95°C. and cooled to 55° C. to generate a cleavage structure. U.S. Pat. No.5,843,669 discloses that a cleavage structure comprises a region of asingle stranded nucleic acid substrate containing secondary structure.Cleavage is carried out in the presence of CleavaseBN nuclease, FEN-1nuclease derived from the archaebacteria Methanococcus jannaschii orboth enzymes. Labeled reaction products are visualized by gelelectrophoresis followed by fluoroimaging. U.S. Pat. No. 5,843,669discloses that CleavaseBN nuclease and Methanococcus jannaschii FEN-1nuclease produce cleavage patterns that are easily distinguished fromeach other, and that the cleavage patterns from a reaction containingboth enzymes include elements of the patterns produced by cleavage witheach individual enzyme but are not merely a composite of the cleavagepatterns produced by each individual enzyme. This indicates that some ofthe fragments that are not cleaved by one enzyme (and which appear as aband in that enzyme's pattern) can be cleaved by a second enzyme in thesame reaction mixture.

Lyamichev et al. disclose a method for detecting DNAs whereinoverlapping pairs of oligonucleotide probes that are partiallycomplementary to a region of target DNA are mixed with the target DNA toform a 5′ flap region, and wherein cleavage of the labeled downstreamprobe by a thermostable FEN-1 nuclease produces a labeled cleavageproduct. Lyamichev et al. also disclose reaction conditions whereinmultiple copies of the downstream oligonucleotide probe can be cleavedfor a single target sequence in the absence of temperature cycling, soas to amplify the cleavage signal and allow quantitative detection oftarget DNA at sub-attomole levels (Lyamichev et al., 1999, Nat.Biotechnol., 17:292).

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science,230:1350.

While the PCR technique is an extremely powerful method for amplifyingnucleic acid sequences, the detection of the amplified material requiresadditional manipulation and subsequent handling of the PCR products todetermine whether the target DNA is present. It is desirable to decreasethe number of subsequent handling steps currently required for thedetection of amplified material. An assay system, wherein a signal isgenerated while the target sequence is amplified, requires fewerhandling steps for the detection of amplified material, as compared to aPCR method that does not generate a signal during the amplificationstep.

U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assay forreleasing labeled probe comprising generating a signal during theamplification step of a PCR reaction in the presence of a nucleic acidto be amplified, Taq polymerase that has 5′ to 3′ exonuclease activityand a 5′, 3′ or 5′ and 3′ end-labeled probe comprising a regioncomplementary to the amplified region and an additionalnon-complementary 5′ tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972disclose further that this PCR based assay can liberate the 5′ labeledend of a hybridized probe when the Taq polymerase is positioned near thelabeled probe by an upstream probe in a polymerization independentmanner, e.g. in the absence of dNTPs.

There is a need in the art for a method of detecting or measuring atarget nucleic acid sequence that does not require multiple steps.

There is also a need in the art for a PCR process for detecting ormeasuring a target nucleic acid sequence that does not require multiplesteps subsequent to the amplification process.

There is also a need in the art for a PCR process for detecting ormeasuring a target nucleic acid sequence that allows for concurrentamplification and detection of a target nucleic acid sequence in asample.

There is also a need in the art for a PCR process for detecting ormeasuring a target nucleic acid sequence wherein the PCR process occursin the presence of a nucleic acid polymerase that lacks 5′ to 3′exonuclease activity.

SUMMARY OF THE INVENTION

The invention provides a method of generating a signal indicative of thepresence of a target nucleic acid sequence in a sample comprisingforming a cleavage structure by incubating a sample comprising a targetnucleic acid sequence with a nucleic acid polymerase, and cleaving thecleavage structure with a FEN nuclease to generate a signal, whereingeneration of the signal is indicative of the presence of a targetnucleic acid sequence in the sample.

As used herein a “FEN nuclease” refers to an enzyme that cleaves acleavage structure according to the invention. The term “FEN nuclease”encompasses an enzyme that consists essentially of a 5′ exonucleaseand/or an endonuclease activity. As used herein, “consists essentiallyof” refers to an enzyme wherein the predominant activity of the enzymeis a 5′ exonucleolytic and/or endonucleolytic activity, such that one orboth of 5′ to 3′ synthetic activity and 3′ single-stranded flap cleavageactivity (i.e., 3′ endonucleolytic and/or 3′exonucleolytic activity) aresubstantially lacking. “Substantially lacks” means that the FEN nucleasepossesses no more than 5% or 10% and preferably less than 0.1%, 0.5%, or1% of the activity of a wild type enzyme (e.g. for 5′ to 3′ syntheticactivity and the 3′ endonucleolytic and/or ′3 exonucleolytic activities,the enzyme may be a wild type DNA polymerase having these activities).5′ to 3′ synthetic activity can be measured, for example, in a nicktranslation assay or an enzymatic sequencing reaction which involve theformation of a phosphodiester bridge between the 3′-hydroxyl group atthe growing end of an oligonucleotide primer and the 5′-phosphate groupof an incoming deoxynucleotide, such that the overall direction ofsynthesis is in the 5′ to 3′ direction. 3′ flap cleavage may be measuredin a DNA synthesis reaction in which, because the (labelled) 3′ end of aDNA duplex is unpaired, it is cleaved from the duplex. A FEN nucleasethat “consists of” a 5′ exonuclease and/or endonuclease activity refersto an enzyme that “lacks” 5′ to 3′ synthetic activity and/or 3′single-stranded flap cleavage activity. “Lacks” means that the Fennuclease has no detectable activity or has only “minor” activity, i.e.,less than 1.0%, 0.5%, 0.1% or 0.01% of the activity of a wild typeenzyme. As used herein, “FEN nuclease” encompasses a 5′ flap-specificnuclease.

As used herein, “wild type” refers to a gene or gene product which hasthe characteristics of (i.e., either has the sequence of or encodes, forthe gene, or possesses the sequence or activity of, for an enzyme) thatgene or gene product when isolated from a naturally occurring source.

A “5′ flap-specific nuclease” (also referred to herein as a“flap-specific nuclease”) according to the invention is an endonucleasewhich can remove a single stranded flap that protrudes as a 5′ singlestrand. A flap-specific nuclease according to the invention can alsocleave a pseudo-Y structure. A substrate of a flap-specific nucleaseaccording to the invention, comprises a target nucleic acid, a secondnucleic acid, a portion of which specifically hybridizes with a targetnucleic acid, and a primer extension product from a third nucleic acidthat specifically hybridizes with a target nucleic acid sequence.

As used herein, a “cleavage structure” refers to a polynucleotidestructure (for example as illustrated in FIG. 1) comprising at least aduplex nucleic acid having a single stranded region comprising a flap, aloop, a single-stranded bubble, a D-loop, a nick or a gap. A cleavagestructure according to the invention thus includes a polynucleotidestructure comprising a flap strand of a branched DNA wherein a 5′single-stranded polynucleotide flap extends from a position near itsjunction to the double stranded portion of the structure and preferablythe flap is labelled with a detectable label. A flap of a cleavagestructure according to the invention is preferably about 1-500nucleotides, more preferably about 5-25 nucleotides and most preferablyabout 10-20 nucleotides and is preferably cleaved at a position locatedeither one nucleotide proximal and/or one nucleotide distal from theelbow of the flap strand.

A cleavage structure according to the invention preferably comprises atarget nucleic acid sequence, and also may include an oligonucleotidethat specifically hybridizes with the target nucleic acid sequence, anda flap extending from the hybridizing oligonucleotide. For example, acleavage structure according to the invention may comprise a targetnucleic acid sequence (for example B in FIG. 3), an upstreamoligonucleotide that is complementary to the target sequence (forexample A in FIG. 3), and a downstream oligonucleotide that iscomplementary to the target sequence (for example C in FIG. 3). In sucha cleavage structure, the downstream oligonucleotide may be blocked atthe 3′ terminus to prevent extension of the 3′ end of the downstreamoligonucleotide.

A cleavage structure according to the invention may be a polynucleotidestructure comprising a flap extending from the downstreamoligonucleotide, wherein the flap is formed by extension of the upstreamoligonucleotide by the synthetic activity of a nucleic acid polymerase,and subsequent, partial, displacement of the 5′ end of the downstreamoligonucleotide.

A cleavage structure according to the invention may be formed byhybridizing a target nucleic acid sequence with an oligonucleotidewherein the oligonucleotide comprises a complementary region thatanneals to the target nucleic acid sequence, and a non-complementaryregion that does not anneal to the target nucleic acid sequence andforms a 5′ flap.

A cleavage structure also may be a pseudo-Y structure wherein a pseudoY-structure is formed if the strand upstream of a flap (referred toherein as a flap adjacent strand or primer strand) is removed, anddouble stranded DNA substrates containing a gap or nick. A “cleavagestructure”, as used herein, does not include a double stranded nucleicacid structure with only a 3′ single-stranded flap. As used herein, a“cleavage structure” comprises ribonucleotides or deoxyribonucleotidesand thus can be RNA or DNA.

A cleavage structure according to the invention may be an overlappingflap wherein the 3′ end of an upstream oligonucleotide capable ofhybridizing to a target nucleic acid sequence (for example A in FIG. 3)is complementary to 1 base pair of the downstream oligonucleotide (forexample C in FIG. 3) that is annealed to a target nucleic acid sequenceand wherein the overlap is directly downstream of the point of extensionof the single stranded flap.

A cleavage structure according to the invention is formed by the stepsof 1. incubating a) an upstream extendable 3′ end, preferably anoligonucleotide primer, b) an oligonucleotide primer probe located notmore than 5000 nucleotides downstream of the upstream primer and c) anappropriate target nucleic acid sequence wherein the target sequence iscomplementary to both the upstream primer and downstream probe and d) asuitable buffer, under conditions that allow the nucleic acid sequenceto hybridize to the oligonucleotide primers, and 2. extending the 3′ endof the upstream oligonucleotide primer by the synthetic activity of apolymerase such that the newly synthesized 3′ end of the upstreamoligonucleotide primer becomes adjacent to and/or displaces at least aportion of (i.e., at least 5-10 nucleotides of) the 5′ end of thedownstream oligonucleotide probe. According to the method of theinvention, buffers and extension temperatures are favorable for stranddisplacement by a particular nucleic acid polymerase according to theinvention. Preferably, the downstream oligonucleotide is blocked at the3′ terminus to prevent extension of the 3′ end of the downstreamoligonucleotide. In another embodiment of the invention, a cleavagestructure according to the invention can be prepared by incubating atarget nucleic acid sequence with an oligonucleotide primer comprising anon-complementary 5′ region that does not anneal to the target nucleicacid sequence and forms a 5′ flap, and a complementary 3′ region thatanneals to the target nucleic acid sequence.

In a preferred embodiment of the invention a cleavage structure islabelled. A labelled cleavage structure according to the invention isformed by the steps of 1. incubating a) an upstream extendable 3′ end,preferably an oligonucleotide primer, b) a labelled probe preferablylocated not more than 5000 and more preferably located not more than 500nucleotides downstream of the upstream primer and c) an appropriatetarget nucleic acid sequence wherein the target sequence iscomplementary to both the primer and the labelled probe and d) asuitable buffer, under conditions that allow the nucleic acid sequenceto hybridize to the primers, and 2. extending the 3′ end of the upstreamprimer by the synthetic activity of a polymerase such that the newlysynthesized 3′ end of the upstream primer partially displaces the 5′ endof the downstream probe. According to the method of the invention,buffers and extension temperatures are favorable for strand displacementby a particular nucleic acid polymerase according to the invention.Preferably, the downstream oligonucleotide is blocked at the 3′ terminusto prevent extension of the 3′ end of the downstream oligonucleotide. Inanother embodiment, a cleavage structure according to the invention canbe prepared by incubating a target nucleic acid sequence with a probecomprising a non-complementary, labeled, 5′ region that does not annealto the target nucleic acid sequence and forms a 5′ flap, and acomplementary 3′ region that anneals to the target nucleic acidsequence.

As used herein, “label” or “labelled moiety capable of providing asignal” refers to any atom or molecule which can be used to provide adetectable (preferably quantifiable) signal, and which can beoperatively linked to a nucleic acid. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, massspectrometry, binding affinity, hybridization radiofrequency and thelike.

As used herein, “generating a signal” refers to detecting and ormeasuring a released nucleic acid fragment as an indication of thepresence of a target nucleic acid sequence in a sample.

As used herein, “sample” refers to any substance containing or presumedto contain a nucleic acid of interest (a target nucleic acid sequence)or which is itself a nucleic acid containing or presumed to contain atarget nucleic acid sequence of interest. The term “sample” thusincludes a sample of nucleic acid (genomic DNA, cDNA, RNA), cell,organism, tissue, fluid, or substance including but not limited to, forexample, plasma, serum, spinal fluid, lymph fluid, synovial fluid,urine, tears, stool, external secretions of the skin, respiratory,intestinal and genitourinary tracts, saliva, blood cells, tumors,organs, tissue, samples of in vitro cell culture constituents, naturalisolates (such as drinking water, seawater, solid materials), microbialspecimens, and objects or specimens that have been “marked” with nucleicacid tracer molecules.

As used herein, “target nucleic acid sequence” or “template nucleic acidsequence” refers to a region of a nucleic acid that is to be eitherreplicated, amplified, and/or detected. In one embodiment, the “targetnucleic acid sequence” or “template nucleic acid sequence” residesbetween two primer sequences used for amplification.

As used herein, “nucleic acid polymerase” refers to an enzyme thatcatalyzes the polymerization of nucleoside triphosphates. Generally, theenzyme will initiate synthesis at the 3′-end of the primer annealed tothe target sequence, and will proceed in the 5′-direction along thetemplate, and if possessing a 5′ to 3′ nuclease activity, hydrolyzingintervening, annealed probe to release both labeled and unlabeled probefragments, until synthesis terminates. Known DNA polymerases include,for example, E. coli DNA polymerase I, T7 DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Bacillus stearothermophilus DNApolymerase, Thermococcus litoralis DNA polymerase, Thermus aquaticus(Taq) DNA polymerase and Pyrococcus furiosus (Pfu) DNA polymerase.

As used herein, “5′ to 3′ exonuclease activity” or “5′→3′ exonucleaseactivity” refers to that activity of a template-specific nucleic acidpolymerase e.g. a 5′→3′ exonuclease activity traditionally associatedwith some DNA polymerases whereby mononucleotides or oligonucleotidesare removed from the 5′ end of a polynucleotide in a sequential manner,(i.e., E. coli DNA polymerase I has this activity whereas the Klenow(Klenow et al., 1970, Proc. Natl. Acad. Sci., USA, 65:168) fragment doesnot, (Klenow et al., 1971, Eur. J. Biochem., 22:371)), orpolynucleotides are removed from the 5′ end by an endonucleolyticactivity that may be inherently present in a 5′ to 3′ exonucleaseactivity.

As used herein, the phrase “substantially lacks 5′ to 3′ exonucleaseactivity” or “substantially lacks 5′-3′ exonuclease activity” meanshaving less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wildtype enzyme. The phrase “lacking 5′ to 3′ exonuclease activity” or“lacking 5′-3′ exonuclease activity” means having undetectable 5′ to 3′exonuclease activity or having less than about 1%, 0.5%, or 0.1% of the5′ to 3′ exonuclease activity of a wild type enzyme. 5′ to 3′exonuclease activity may be measured by an exonuclease assay whichincludes the steps of cleaving a nicked substrate in the presence of anappropriate buffer, for example 10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂ and50 μg/ml bovine serum albumin) for 30 minutes at 60° C., terminating thecleavage reaction by the addition of 95% formamide containing 10 mM EDTAand 1 mg/ml bromophenol blue, and detecting nicked or un-nicked product.

Nucleic acid polymerases useful according to the invention include butare not limited to Pfu, exo-Pfu (a mutant form of Pfu that lacks 3′ to5′ exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst,N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form ofVent that lacks 3′ to 5′ exonuclease activity), Deep Vent, Deep Ventexo- (a mutant form of Deep Vent that lacks 3′ to 5′ exonucleaseactivity), U1Tma and Sequenase. Additional nucleic acid polymerasesuseful according to the invention are included below in the sectionentitled, “Nucleic Acid Polymerases”.

As used herein, “cleaving” refers to enzymatically separating a cleavagestructure into distinct (i.e. not physically linked to other fragmentsor nucleic acids by phosphodiester bonds) fragments or nucleotides andfragments that are released from the cleavage structure. For example,cleaving a labelled cleavage structure refers to separating a labelledcleavage structure according to the invention and defined below, intodistinct fragments including fragments derived from an oligonucleotidethat specifically hybridizes with a target nucleic acid sequence orwherein one of the distinct fragments is a labelled nucleic acidfragment derived from a target nucleic acid sequence and/or derived froman oligonucleotide that specifically hybridizes with a target nucleicacid sequence that can be detected and/or measured by methods well knownin the art and described herein that are suitable for detecting thelabelled moiety that is present on a labelled fragment.

As used herein, “endonuclease” refers to an enzyme that cleaves bonds,preferably phosphodiester bonds, within a nucleic acid molecule. Anendonuclease according to the invention can be specific forsingle-stranded or double-stranded DNA or RNA.

As used herein, “exonuclease” refers to an enzyme that cleaves bonds,preferably phosphodiester bonds, between nucleotides one at a time fromthe end of a polynucleotide. An exonuclease according to the inventioncan be specific for the 5′ or 3′ end of a DNA or RNA molecule, and isreferred to herein as a 5′ exonuclease or a 3′ exonuclease.

As used herein a “flap” refers to a region of single stranded DNA thatextends from a double stranded nucleic acid molecule. A flap accordingto the invention is preferably between about 1-500 nucleotides, morepreferably between about 5-25 nucleotides and most preferably betweenabout 10-20 nucleotides.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

In a preferred embodiment, the cleavage structure comprises at least oneoligonucleotide primer.

The invention also provides a method of detecting or measuring a targetnucleic acid sequence comprising forming a cleavage structure byincubating a sample comprising a target nucleic acid sequence with anucleic acid polymerase, cleaving the cleavage structure with a FENnuclease to release a nucleic acid fragment, and detecting and/ormeasuring the release of the fragment as an indication of the presenceof the target sequence in the sample.

As used herein, “detecting a target nucleic acid sequence” or “measuringa target nucleic acid sequence” refers to determining the presence of aparticular target nucleic acid sequence in a sample or determining theamount of a particular target nucleic acid sequence in a sample as anindication of the presence of a target nucleic acid sequence in asample. The amount of a target nucleic acid sequence that can bemeasured or detected is preferably about 1 molecule to 10²⁰ molecules,more preferably about 100 molecules to 10¹⁷ molecules and mostpreferably about 1000 molecules to 10¹⁴ molecules. According to theinvention, the detected nucleic acid is derived from the labelled 5′ endof a downstream probe of a cleavage structure according to the invention(for example C in FIG. 3), that is displaced from the target nucleicacid sequence by the 3′ extension of an upstream probe of a cleavagestructure according to the invention (for example A of FIG. 3).According to the present invention, a label is attached to the 5′ end ofthe downstream probe (for example C in FIG. 3) comprising a cleavagestructure according to the invention. Alternatively, a label is attachedto the 3′ end of the downstream probe and a quencher is attached to the5′ flap of the downstream probe. According to the invention, a label maybe attached to the 3′ end of the downstream probe (for example C in FIG.3) comprising a cleavage structure according to the invention.

According to the invention, the downstream probe (for example C in FIG.3) may be labelled internally. In a preferred embodiment, a cleavagestructure according to the invention can be prepared by incubating atarget nucleic acid sequence with a probe comprising anon-complementary, labeled, 5′ region that does not anneal to the targetnucleic acid sequence and forms a 5′ flap, and a complementary 3′ regionthat anneals to the target nucleic acid sequence. According to thisembodiment of the invention, the detected nucleic acid is derived fromthe labelled 5′ flap region of the probe. Preferably there is a directcorrelation between the amount of the target nucleic acid sequence andthe signal generated by the cleaved, detected nucleic acid.

As used herein, “detecting release of labelled fragments” or “measuringrelease of labelled fragments” refers to determining the presence of alabelled fragment in a sample or determining the amount of a labelledfragment in a sample. Methods well known in the art and described hereincan be used to detect or measure release of labelled fragments. A methodof detecting or measuring release of labelled fragments will beappropriate for measuring or detecting the labelled moiety that ispresent on the labelled fragments. The amount of a released labelledfragment that can be measured or detected is preferably about 25%, morepreferably about 50% and most preferably about 95% of the total startingamount of labelled probe.

As used herein, “labelled fragments” refer to cleaved mononucleotides orsmall oligonucleotides or oligonucleotides derived from the labelledcleavage structure according to the invention wherein the cleavedoligonucleotides are preferably between about 2-1000 nucleotides, morepreferably between about 5-50 nucleotides and most preferably betweenabout 16-18 nucleotides, which are cleaved from a cleavage structure bya FEN nuclease and can be detected by methods well known in the art anddescribed herein.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

In another preferred embodiment, the nucleic acid polymerase is a DNApolymerase.

In another preferred embodiment, the nucleic acid polymerase isthermostable.

As used herein, “thermostable” refers to an enzyme which is stable andactive at temperatures as great as preferably between about 90-100° C.and more preferably between about 70-98° C. to heat as compared, forexample, to a non-thermostable form of an enzyme with a similaractivity. For example, a thermostable nucleic acid polymerase or FENnuclease derived from thermophilic organisms such as P. furiosus, M.jannaschii, A. fulgidus or P. horikoshii are more stable and active atelevated temperatures as compared to a nucleic acid polymerase from E.coli or a mammalian FEN enzyme. A representative thermostable nucleicacid polymerase isolated from Thermus aquaticus (Taq) is described inU.S. Pat. No. 4,889,818 and a method for using it in conventional PCR isdescribed in Saiki et al., 1988, Science 239:487. Another representativethermostable nucleic acid polymerase isolated from P. furiosus (Pfu) isdescribed in Lundberg et al., 1991, Gene, 108:1-6. Additionalrepresentative temperature stable polymerases include, e.g., polymerasesextracted from the thermophilic bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, or from thermophilic archaeaThermococcus litoralis, and Methanothermus fervidus.

Temperature stable polymerases and FEN nucleases are preferred in athermocycling process wherein double stranded nucleic acids aredenatured by exposure to a high temperature (about 95° C.) during thePCR cycle.

In another preferred embodiment, the FEN nuclease is a flap-specificnuclease.

In another preferred embodiment, the FEN nuclease is thermostable.

In another preferred embodiment, the cleavage structure is formedcomprising at least one labeled moiety capable of providing a signal.

In another preferred embodiment, the cleavage structure is formedcomprising a pair of interactive signal generating labeled moietieseffectively positioned to quench the generation of a detectable signal,wherein the labeled moieties are separated by a site susceptible to FENnuclease cleavage, thereby allowing the nuclease activity of the FENnuclease to separate the first interactive signal generating labeledmoiety from the second interactive signal generating labeled moiety bycleaving at the site susceptible to FEN nuclease, thereby generating adetectable signal.

In another preferred embodiment, the pair of interactive signalgenerating moieties comprises a quencher moiety and a fluorescentmoiety.

In another preferred embodiment, the cleavage structure comprises atleast one oligonucleotide primer.

The invention also provides a polymerase chain reaction process fordetecting a target nucleic acid sequence in a sample comprisingproviding a cleavage structure, providing a set of oligonucleotideprimers wherein a first primer contains a sequence complementary to aregion in one strand of the target nucleic acid sequence and primes thesynthesis of a complementary DNA strand, and a second primer contains asequence complementary to a region in a second strand of the targetnucleic acid sequence and primes the synthesis of a complementary DNAstrand, and amplifying the target nucleic acid sequence employing anucleic acid polymerase as a template-dependent polymerizing agent underconditions which are permissive for PCR cycling steps of (i) annealingof primers required for amplification to a template nucleic acidsequence contained within the target sequence and annealing primersrequired for formation of a cleavage structure to a target nucleic acidsequence, (ii) extending the primers wherein the nucleic acid polymerasesynthesizes a primer extension product, and (iii) cleaving the cleavagestructure employing a FEN nuclease as a cleavage agent for release oflabeled fragments from the cleavage structure thereby creatingdetectable labeled fragments; and (d) detecting and/or measuring therelease of labeled fragments as an indication of the presence of thetarget nucleic acid sequence in the sample.

The invention provides for a polymerase chain reaction process whereinamplification and detection of a target nucleic acid sequence occurconcurrently (i.e. real time detection). The invention also provides fora polymerase chain reaction process wherein amplification of a targetnucleic acid sequence occurs prior to detection of the target nucleicacid sequence (i.e. end point detection).

As used herein, an “oligonucleotide primer” refers to a single strandedDNA or RNA molecule that is hybridizable to a nucleic acid template andprimes enzymatic synthesis of a second nucleic acid strand.Oligonucleotide primers useful according to the invention are betweenabout 10 to 100 nucleotides in length, preferably about 17-50nucleotides in length and more preferably about 17-45 nucleotides inlength. Oligonucleotide probes useful for the formation of a cleavagestructure according to the invention are between about 17-40 nucleotidesin length, preferably about 17-30 nucleotides in length and morepreferably about 17-25 nucleotides in length.

As used herein, “template dependent polymerizing agent” refers to anenzyme capable of extending an oligonucleotide primer in the presence ofadequate amounts of the four deoxyribonucleoside triphosphates (dATP,dGTP, dCTP and dTTP) or analogs as described herein, in a reactionmedium comprising appropriate salts, metal cations, appropriatestabilizers and a pH buffering system. Template dependent polymerizingagents are enzymes known to catalyze primer- and template-dependent DNAsynthesis, and possess 5′ to 3′ nuclease activity. Preferably, atemplate dependent polymerizing agent according to the invention lacks5′ to 3′ nuclease activity.

As used herein, “amplifying” refers to producing additional copies of anucleic acid sequence, including the method of the polymerase chainreaction.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

In a preferred embodiment, the oligonucleotide primers of step b of thepolymerase chain reaction process described above are oriented such thatthe forward primer is located upstream of a cleavage structure accordingto the invention and the reverse primer is located downstream of acleavage structure according to the invention. The reverse primer iscomplementary to the opposite strand of the forward primer which iscomplementary to a strand of the cleavage structure.

In another preferred embodiment, the nucleic acid polymerase is a DNApolymerase.

In another preferred embodiment, the nucleic acid polymerase isthermostable.

In another preferred embodiment, the nucleic acid polymerase is selectedfrom the group consisting of Taq polymerase and Pfu polymerase.

In another preferred embodiment the FEN nuclease is thermostable.

In another preferred embodiment the FEN nuclease is a flap-specificnuclease.

In another preferred embodiment the FEN nuclease is selected from thegroup consisting of FEN nuclease enzyme derived from Archaeglobusfulgidus, Methanococcus jannaschii, Pyrococcus furiosus, human, mouse orXenopus laevis. A FEN nuclease according to the invention also includesSaccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, PolI DNA polymerase associated 5′ to 3′ exonuclease domain, (e.g. E. coli,Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax(Bca), Streptococcus pneumoniae) and phage functional homologs of FENincluding but not limited to T4 RNaseH, T5 5′ to 3′ exonuclease, T7 gene6 exonuclease and T3 gene 6 exonuclease.

Preferably, only the 5′ to 3′ exonuclease domains of Taq, Tfl and BcaFEN nuclease are used.

In another preferred embodiment, the labeled cleavage structure isformed by the addition of at least one labeled moiety capable ofproviding a signal.

The invention also provides a polymerase chain reaction process forsimultaneously forming a cleavage structure, amplifying a target nucleicacid sequence in a sample and cleaving the cleavage structurecomprising: (a) providing an upstream oligonucleotide primercomplementary to a region in one strand of the target nucleic acidsequence and a downstream labeled probe complementary to a region in thesame strand of the target nucleic acid sequence, wherein the upstreamprimer contains a sequence complementary to a region in one strand ofthe target nucleic acid sequence and primes the synthesis of acomplementary DNA strand, and the downstream probe contains a sequencecomplementary to a region in a second strand of the target nucleic acidsequence and primes the synthesis of a complementary DNA strand; and (b)detecting a nucleic acid which is produced in a reaction comprisingamplification of the target nucleic acid sequence and cleavage thereofwherein a nucleic acid polymerase is a template-dependent polymerizingagent under conditions which are permissive for PCR cycling steps of (i)annealing of primers to a target nucleic acid sequence, (ii) extendingthe primers of step (a) wherein the nucleic acid polymerase synthesizesprimer extension products, and wherein the primer extension product ofthe primer of step (a) partially displaces the downstream probe of step(a) to form a cleavage structure; and (iii) cleaving the cleavagestructure employing a FEN nuclease as a cleavage agent for release oflabeled fragments from the cleavage structure thereby creatingdetectable labeled fragments.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

The invention also provides a method of forming a cleavage structurecomprising the steps of: (a) providing a target nucleic acid sequence,(b) providing an upstream primer complementary to said target nucleicacid sequence, (c) providing a downstream probe complementary to saidtarget nucleic acid sequence, (d) extending the 3′ end of the upstreamprimer with a nucleic acid polymerase; and (e) displacing the 5′ end ofthe downstream probe.

Preferably the downstream probe is located not more than 500 nucleotidesdownstream of the upstream primer.

During the extension step, the 3′ end of the upstream primer is extendedby the synthetic activity of a polymerase such that the newlysynthesized 3′ end of the upstream primer partially displaces the 5′ endof the downstream probe.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

In another embodiment of the invention, a cleavage structure accordingto the invention can be prepared by incubating a target nucleic acidsequence with a probe comprising a non-complementary 5′ region that doesnot anneal to the target nucleic acid sequence and forms a 5′ flap, anda complementary 3′ region that anneals to the target nucleic acidsequence.

The invention also provides a method of forming a labeled cleavagestructure comprising the steps of: (a) providing a target nucleic acidsequence, (b) providing an upstream primer complementary to said targetnucleic acid sequence, (c) providing a downstream end labeled probecomplementary to said target nucleic acid sequence, (d) extending the 3′end of the upstream primer with a nucleic acid polymerase; and (e)displacing the 5′ end of the downstream probe.

Preferably the downstream end labeled probe is located not more than 500nucleotides downstream of the upstream primer. Preferably, thedownstream oligonucleotide is blocked at the 3′ terminus to preventextension of the 3′ end of the downstream oligonucleotide. Such blockagecan be achieved by placing a phosphate, or other moiety not readilyremoved, on the 3′ terminal hydroxyl of the oligonucleotide.

During the extension step, the 3′ end of the upstream primer is extendedby the synthetic activity of a polymerase such that the newlysynthesized 3′ end of the upstream primer partially displaces the 5′ endof the downstream probe. According to the method of the invention,buffers and extension temperatures are favorable for strand displacementby a particular nucleic acid polymerase according to the invention.

In one embodiment of the invention, a cleavage structure according tothe invention can be prepared by incubating a target nucleic acidsequence with a probe comprising a non-complementary, labeled, 5′ regionthat does not anneal to the target nucleic acid sequence and forms a 5′flap, and a complementary 3′ region that anneals to the target nucleicacid sequence.

The invention also provides a kit for generating a signal indicative ofthe presence of a target nucleic acid sequence in a sample comprising anucleic acid polymerase, a FEN nuclease and a suitable buffer. In apreferred embodiment, the invention also provides a kit for generating asignal indicative of the presence of a target nucleic acid sequence in asample comprising one or more nucleic acid polymerases, a FEN nucleaseand a suitable buffer.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks 5′ to 3′ exonuclease activity.

In a preferred embodiment the nucleic acid polymerase is thermostable.

In another preferred embodiment the FEN nuclease is thermostable.

In another preferred embodiment the kit further comprises a labelednucleic acid complementary to the target nucleic acid sequence.

The invention also provides a composition comprising a nucleic acidpolymerase and a FEN nuclease.

In a preferred embodiment, the nucleic acid polymerase substantiallylacks a 5′ to 3′ exonuclease activity.

In another preferred embodiment the invention provides for a compositioncomprising one or more nucleic acid polymerases and a FEN nuclease.

Further features and advantages of the invention are as follows. Theclaimed invention provides a method of generating a signal to detectand/or measure a target nucleic acid wherein the generation of a signalis an indication of the presence of a target nucleic acid in a sample.The method of the claimed invention does not require multiple steps. Theclaimed invention also provides a PCR based method for detecting and/ormeasuring a target nucleic acid comprising generating a signal as anindication of the presence of a target nucleic acid. The claimedinvention allows for simultaneous amplification and detection and/ormeasurement of a target nucleic acid sequence. The claimed inventionalso provides a PCR based method for detecting and/or measuring a targetnucleic acid comprising generating a signal in the absence of a nucleicacid polymerase that demonstrates 5′ to 3′ exonuclease activity.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 demonstrates FEN nuclease cleavage structures.

FIG. 2 demonstrates three templates (labeled 1, 2, and 3) that may beused to detect FEN nuclease activity.

FIG. 3 is a diagram illustrating a synthesis and cleavage reaction togenerate a signal according to the invention.

FIG. 4 is a Sypro Orange stained polyacrylamide gel demonstratingCBP-tagged Pfu FEN-1 protein.

FIG. 5 is an autoradiograph of a FEN-1 nuclease assay.

FIG. 6 is a graph representing detection of β-actin sequences in genomicDNA using fluorescently labeled β-actin probe in the presence of FEN-1and a Taq polymerase deficient in 5′ to 3′ exonuclease activity.

FIG. 7 is a graph representing detection of β-actin sequences in genomicDNA using fluorescently labeled β-actin probe in the presence of FEN-1and a Pfu polymerase deficient in 3′ to 5′ exonuclease activity.

FIG. 8 is a representation of an open circle probe for rolling circleamplification.

FIG. 9 is a representation of rolling circle amplification.

FIG. 10 is a representation of a safety pin probe.

FIG. 11 is a representation of a scorpion probe.

FIG. 12 is a representation of a sunrise/amplifluor probe.

DESCRIPTION

The invention provides for a method of generating a signal to detect thepresence of a target nucleic acid in a sample wherein a nucleic acid istreated with the combination of a nucleic acid polymerase and a FENnuclease. The invention also provides for a process for detecting ormeasuring a nucleic acid that allows for concurrent amplification,cleavage and detection of a target nucleic acid sequence in a sample.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A LaboratoryManual, Second Edition; Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Nucleic Acid Hybridization (B. D. Harnes & S. J. Higgins, eds.,1984); A Practical Guide to Molecular Cloning (B. Perbal, 1984); and aseries, Methods in Enzymology (Academic Press, Inc.). All patents,patent applications, and publications mentioned herein, both supra andinfra, are hereby incorporated by reference.

I. FEN Nucleases

FEN-1 is an ˜40 kDa divalent metal ion-dependent exo- and endonucleasethat specifically recognizes the backbone of a 5′ single-stranded flapstrand and tracks down this arm to the cleavage site, which is locatedat the junction wherein the two strands of duplex DNA adjoin thesingle-stranded arm. Both the endo- and exonucleolytic activities showlittle sensitivity to the base at the most 5′ position at the flap ornick. Both FEN-1 endo- and exonucleolytic substrate binding and cuttingare stimulated by an upstream oligonucleotide (flap adjacent strand orprimer). This is also the case for E. coli pol I. The endonucleaseactivity of the enzyme is independent of the 5′ flap length, cleaving a5′ flap as small as one nucleotide. The endonuclease and exonucleaseactivities are insensitive to the chemical nature of the substrate,cleaving both DNA and RNA.

Both the endo- and exonucleolytic activities are inhibited byconcentrations of salts in the physiological range. The exonucleaseactivity is inhibited 50-fold at 50 mM NaCl as compared to 0 mM NaCl.The endonuclease activity is inhibited only sevenfold at 50 mM NaCl(Reviewed in Lieber 1997, supra).

Although a 5′-OH terminus is a good substrate for FEN-1 loading onto a5′ flap substrate, it serves as a very poor substrate when part of anick in an otherwise double stranded DNA structure. The electrostaticrepulsion by the terminal phosphate is likely to favor breathing of thesubstrate into a pseudo-flap configuration, providing the active form ofthe substrate for FEN-1. Such an explanation would indicate a singleactive site and a single mechanism of loading of FEN-1 onto the 5′ ssDNAterminus of the flap or pseudo-flap configuration of the nick.Consistent with this model are observations that optimal activity at anick requires very low Mg²⁺ and monovalent salt concentrations, whichdestabilize base-pairing and would favor breathing of a nick to a flap.Higher Mg²⁺ and monovalent salt concentrations would disfavor breathingand inhibit cutting of nicked or gapped structures that do requirebreathing to convert to a flap. Cleavage of stable flap structures isoptimal at moderate Mg²⁺ levels and does not decrease with increasingMg²⁺ concentration. This is because a flap substrate does not have tomelt out base pairs to achieve its structure; hence, it is entirelyinsensitive to Mg²⁺. Though the endonucleolytic activity decreases withmonovalent salt, the decline is not nearly as sharp as that seen for theexonucleolytic activity. Furthermore, it has previously been shown thatone-nucleotide flaps are efficient substrates. All of these observationsare consistent with the fact that when FEN-1 has been interpreted to befunctioning as an exonuclease, the size of the degradation products varyfrom one to several nucleotides in length. Breathing of nicks into flapsof varying length would be expected to vary with local sequence,depending on the G/C content. In summary, a nick breathing to form atransient flap means that the exonucleolytic activity of FEN-1 is thesame as the endonucleolytic activity (Reviewed in Lieber, 1997, supra).

The endonuclease and exonuclease activities of FEN-1 cleave both DNA andRNA without requiring accessory proteins. At the replication fork,however, FEN-1 does interact with other proteins, including a DNAhelicase and the proliferating cell nuclear antigen (PCNA), theprocessivity factor for DNA polymerases δ and ε. PCNA significantlystimulates FEN-1 endo- and exonucleolytic activity.

The FEN-1 enzymes are functionally related to several smallerbacteriophage 5′→3′ exonucleases such as T5 5′ exonuclease and T4 RNaseH as well as to the larger eukaryotic nucleotide excision repair enzymessuch as XPG, which also acts in the transcription-coupled repair ofoxidative base damage. In eubacteria such as Escherichia coli andThermus aquaticus, Okazaki processing is provided by the PolI 5′→3′exonuclease domain. These bacterial and phage enzymes share two areas oflimited sequence homology with FEN-1, which are termed the N(N-terminal)and I (intermediate) regions, with the residue similarities concentratedaround seven conserved acidic residues. Based on crystal structures ofT4 RNase H and T5 exonuclease as well as mutagenesis data, it has beenproposed that these residues bind to two Mg²⁺ ions that are required foraffecting DNA hydrolysis; however, the role each metal plays in thecatalytic cycle, which is subtly different for each enzyme, is not wellunderstood (Reviewed in Hosfield et al., 1998b, supra).

fen-1 genes encoding FEN-1 enzymes useful in the invention includemurine fen-1, human fen-1, rat fen-1, Xenopus laevis fen-1, and fen-1genes derived from four archaebacteria Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus and Pyrococcus horikoshii.cDNA clones encoding FEN-1 enzymes have been isolated from human(GenBank Accession Nos.: NM_(—)004111 and L37374), mouse (GenBankAccession No.: L26320), rat (GenBank Accession No.: AA819793), Xenopuslaevis (GenBank Accession Nos.: U68141 and U64563), and P. furiosus(GenBank Accession No.: AF013497). The complete nucleotide sequence forP. horikoshii flap endonuclease has also been determined (GenBankAccession No.: AB005215). The FEN-1 family also includes theSaccharomyces cerevisiae RAD27 gene (GenBank Accession No.: Z28113Y13137) and the Saccharomyces pombe RAD2 gene (GenBank Accession No.:X77041). The archaeal genome of Methanobacterium thermautotrophiculumhas also been sequenced. Although the sequence similarity between FEN-1and prokaryotic and viral 5′→3′ exonucleases is low, FEN-1s within theeukaryotic kingdom are highly conserved at the amino acid level, withthe human and S. cerevisiae proteins being 60% identical and 78%similar. The three archaebacterial FEN-1 proteins are also, highlyhomologous to the eukaryotic FEN-1 enzymes (Reviewed in Matsui et al.,1999., J. Biol. Chem., 274:18297, Hosfield et al., 1998b, J. Biol.Chem., 273:27154 and Lieber, 1997, BioEssays, 19:233).

The sequence similarities in the two conserved nuclease domains(N-terminal or N and intermediate or I domains) between human and otherFEN-1 family members are 92% (murine), 79% (S. cerevisiae), 77% (S.pombe), 72% (A. fulgidus), 76% (M. jannaschii), and 74% (P. furiosus).

FEN-1 specifically recognizes the backbone of a 5′ single-stranded flapstrand and migrates down this flap arm to the cleavage site located atthe junction between the two strands of duplex DNA and thesingle-stranded arm. If the strand upstream of the flap (sometimescalled the flap adjacent strand or primer strand) is removed, theresulting structure is termed a pseudo-Y (see FIG. 1). This structure iscleaved by FEN-1, but at 20- to 100-fold lower efficiency. FEN-1 doesnot cleave 3′ single-stranded flaps. However, FEN-1 acting as anexonuclease will hydrolyze dsDNA substrates containing a gap or nick(Reviewed in Hosfield et al., 1998a, supra, Hosfield et al., 1999b,supra and Lieber 1997, supra). Exonucleolytically, FEN-1 acts at a nickand, with lower efficiency, at a gap or a recessed 5′ end on dsDNA. Atgapped structures, the efficiency of FEN-1 binding and cutting decreaseswith increasing gap size up to approximately five nucleotides and thenstabilizes at a level of cleavage that is equivalent to activity on arecessed 5′ end within dsDNA. Blunt dsDNA, recessed 3′ ends and ssDNAare not cleaved (Reviewed in Lieber 1997, supra).

FEN nucleases that are useful according to the invention have beenisolated from a variety of organisms including human (GenBank AccessionNos.: NM_(—)004111 and L37374), mouse (GenBank Accession No.: L26320),rat (GenBank Accession No.: AA819793), yeast (GenBank Accession No.:Z28113 Y13137 and GenBank Accession No.: X77041) and xenopus laevis(GenBank Accession Nos.: U68141 and U64563). Such enzymes can be clonedand overexpressed using conventional techniques well known in the art.

A FEN nuclease according to the invention is preferably thermostable.Thermostable FEN nucleases have been isolated and characterized from avariety of thermostable organisms including four archeaebacteria. ThecDNA sequence (GenBank Accession No.: AF013497) and the amino acidsequence (Hosfield et al., 1998a, supra and Hosfield et al., 1998b) forP. furiosus flap endonuclease have been determined. The completenucleotide sequence (GenBank Accession No.: AB005215) and the amino acidsequence (Matsui et al., supra) for P. horikoshii flap endonuclease havealso been determined. The amino acid sequence for M. jannaschii(Hosfield et al., 1998b and Matsui et al., 1999 supra) and A. fulgidus(Hosfield et al., 1998b) flap endonuclease have also been determined.

Thermostable FEN1 enzymes can be cloned and overexpressed usingtechniques well known in the art and described in Hosfield et al.,1998a, supra, Hosfield et al., 1998b, Kaiser et al., 1999, J. Biol.Chem., 274: 21387 and Matusi et al., supra and herein in Example 2entitled “Cloning Pfu FEN-1”.

The endonuclease activity of a FEN enzyme can be measured by a varietyof methods including the following.

A. FEN Endonuclease Activity Assay

1. Templates (for example as shown in FIG. 2) are used to evaluate theactivity of a FEN nuclease according to the invention.

Template 1 is a 5′ ³³P labeled oligonucleotide (Heltest4) with thefollowing sequence:

(SEQ ID NO: 1) 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG 3′.The underlined section of Heltest4 represents the region complementaryto M13mp18+. The cleavage product is an 18 nucleotide fragment with thesequence AAAATAAATAAAAAAAAT (SEQ ID NO: 2).

Heltest4 binds to M13 to produce a complementary double stranded domainas well as a non-complementary 5′ overhang. This duplex forms template 2(FIG. 2) which is also used for helicase assays. Template 3 (FIG. 2) hasan additional primer (FENAS) bound to M13 and is directly adjacent toHeltest 4. The sequence of FENAS is: 5′ CCATTCGCCATTCAGGCTGCGCA 3′ (SEQID NO: 3). In the presence of template 3, FEN binds the free 5′ terminusof Heltest4, migrates to the junction and cleaves Heltest4 to produce an18 nucleotide fragment. Templates 1 and 2 serve as controls, althoughtemplate 2 can also serve as a template.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl 

10×Pfu buffer is available from Stratagene (Catalog #200536). Accordingto the method of the invention, 10×Pfu buffer is diluted such that areaction is carried out in the presence of 1× buffer.

M13 is M13mp18+ strand and is at a concentration of 200 ng/μL, ³³Plabeled Heltest4 is at an approximate concentration of 0.7 ng/μl, andFENAS is at a concentration of 4.3 ng/μl. Based on these concentrations,the Heltest4 and M13 are at approximately equal molar amounts (5×10⁻¹⁴)and FENAS is present in an approximately 10× molar excess (6×10⁻¹³).

The template mixture is heated at 950 C for five minutes, cooled to roomtemperature for 45 minutes and stored at 4° C. overnight.

2 μl of FEN-1 or, as a control, H₂O are mixed with the three templatesas follows:

  3 μl template  0.7 μl 10x cloned Pfu buffer 0.56 μl 100 mM MgCl₂ 2.00μl enzyme or H₂O 0.74 μl H₂0 7.00 μl total volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide, 7M urea CastAway (Stratagene) gel.

Alternatively, FEN activity can be analyzed in the following bufferwherein a one hour incubation time is utilized.

10×FEN Buffer

500 mM Tris-HCl pH 8.0

100 mM MgCl₂

The reaction mixture below is mixed with 2 μl of FEN or, as a control,211 of H₂O.

  3 μl template  0.7 μl 10x FEN buffer 2.00 μl enzyme or H₂O  1.3 μl H₂O7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution, samples are heated at 99° C. for five minutes. Samples areloaded on an eleven-inch long, hand-poured, 20% acrylamide/bisacrylamide, 7M urea gel. The gel is run at 20 watts until thebromophenol blue has migrated approximately ⅔ the total distance. Thegel is removed from the glass plates and soaked for 10 minutes in fix(15% methanol, 5% acetic acid) and then for 10 minutes in water. The gelis placed on Whatmann 3 mm paper, covered with plastic wrap and driedfor 2 hours in a heated vacuum gel dryer. The gel is exposed overnightto X-ray film.

2. FEN endonuclease activity can also be measured according to themethod of Kaiser et al., supra). Briefly, reactions are carried out in a10 μl volume containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05%Nonidet P-40, 10 μg/ml tRNA, and 200 mM KCl for TaqPol and TthPol or 50mM KCl for all other enzymes. Reaction conditions can be varieddepending on the cleavage structure being analyzed. Substrates (2 μM)and varying amounts of enzyme are mixed with the indicated (above)reaction buffer and overlaid with Chill-out (MJ Research) liquid wax.Substrates are heat denatured at 90° C. for 20 s and cooled to 50° C.,then reactions are started by addition of MgCl₂ or MnCl₂ and incubatedat 500 C for the specified length of time. Reactions are stopped by theaddition of 10 μl of 95% formamide containing 10 mM EDTA and 0.02%methyl violet (Sigma). Samples are heated to 90° C. for 1 minimmediately before electrophoresis on a 20% denaturing acrylamide gel(19:1 cross-linked), with 7M urea, and in a buffer of 45 mM Tris borate,pH 8.3, 1.4 mM EDTA. Unless otherwise indicated, 1 μl of each stoppedreaction is loaded per lane. Gels are scanned on an FMBIO-100fluorescent gel scanner (Hitachi) using a 505-nm filter. The fraction ofcleaved product is determined from intensities of bands corresponding touncut and cut substrate with FMBIO Analysis software (version 6.0,Hitachi). The fraction of cut product should not exceed 20% to ensurethat measurements approximate initial cleavage rates. The cleavage rateis defined as the concentration of cut product divided by the enzymeconcentration and the time of the reaction (in minutes). For each enzymethree data points are used to determine the rate and experimental error.

3. FEN endonuclease activity can also be measured according to themethod of Hosfield et al., 1998a, supra. Briefly, in a final volume of13 μl, varying amounts of FEN and 1.54 pmol of labeled cleavagesubstrate are incubated at different temperatures for 30 min before thereaction is quenched with an equal volume of stop solution (10 mM EDTA,95% deionized formamide, and 0.008% bromophenol blue and xylene cyanol).Samples are electrophoresed through denaturing 15% polyacrylamide gels,and the relative amounts of starting material and product arequantitated using the IPLabGel system (Stratagene) running MacBAS imageanalysis software. Most reactions are performed in standard assay buffer(10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 50 μg/ml bovine serumalbumin); however, in a series of experiments the effect of differentdivalent metals and pH levels are studied by varying the standardbuffer. For divalent metals, MgCl₂ is omitted, and different metal ionsare used at a final concentration of 10 mM. To study the influence ofpH, buffers containing different amounts of Tris-HCl, glycine, andsodium acetate are used at a final concentration of 10 mM to obtain awide range of pH levels at 25° C.

4. FEN endonuclease activity can also be measured according to themethod of Matusi et al., 1999, supra. Briefly, the enzyme are performedin a 15-μl reaction mixture containing 50 mM Tris-HCl (pH 7.4), 1.5 mMMgCl₂, 0.5 mM β-mercaptoethanol, 100 μg/ml bovine serum albumin, and 0.6pmol of a labeled cleavage structure. After incubation for 30 min at 60°C., the reaction is terminated by adding 15 μl of 95% formamidecontaining 10 mM EDTA and 1 mg/ml bromphenol blue. The samples areheated at 95° C. for 10 min, loaded onto a 15% polyacrylamide gel (35cm×42.5 cm) containing 7M urea and 10×TBE (89 mM Tris-HCl, 89 mM boricacid, 2 mM EDTA (ph 8.0)), and then electrophoresed for 2 h at 2000 V.Reaction products are visualized and quantified using a PhosphorImager(Bio-Rad). Size marker, oligonucleotides are 5′ end-labeled with[γ-³²P]ATP and T4 polynucleotide kinase.

To determine the optimum pH, the reaction is performed in an assaymixture (15 μl) containing 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100μg/ml bovine serum albumin, and 0.6 pmol of 5′ end-labeled cleavagestructure in 50 mM of one of the following buffers at 60° C. for 30 min.Three different 50 mM buffers are used to obtain a wide pH range asfollows: sodium acetate buffer (pH 4.0-5.5), phosphate buffer (pH5.5-8.0), and borate buffer (pH 8.0-9.4).

B. FEN Exonuclease Activity Assay

The exonuclease activity of a FEN nuclease according to the inventioncan be measured by the method of measuring FEN-1 endonuclease activitydescribed in Matsui et al., 1999, supra and summarized above.

Alternatively, the exonuclease activity of a FEN enzyme can be analyzedby the method described in Hosfield et al., 1998b, supra. Briefly,exonuclease activities are assayed using a nicked substrate of FEN underconditions identical to those described for the endonuclease assays(described above).

The precise positions of DNA cleavage in both the exonuclease andendonuclease experiments can be obtained by partial digestion of a 5′³²P-labeled template strand using the 3′-5′ exonuclease activity ofKlenow fragment.

A cleavage structure according to the invention comprises a partiallydisplaced 5′ end of an oligonucleotide annealed to a target nucleic acidsequence. Another cleavage structure according to the inventioncomprises a target nucleic acid sequence (for example B in FIG. 3), anupstream oligonucleotide that is complementary to the target sequence(for example A in FIG. 3), and a downstream oligonucleotide that iscomplementary to the target sequence (for example C in FIG. 3). Acleavage structure according to the invention can be formed by overlapbetween the upstream oligonucleotide and the downstream probe, or byextension of the upstream oligonucleotide by the synthetic activity of anucleic acid polymerase, and subsequent partial displacement of the 5′end of the downstream oligonucleotide. A cleavage structure of this typeis formed according to the method described in the section entitled“Cleavage Structure”.

Alternatively, a cleavage structure according to the invention is formedby annealing a target nucleic acid sequence to an oligonucleotidewherein the oligonucleotide comprises a complementary region thatanneals to the target nucleic acid sequence, and a non-complementaryregion that does not anneal to the target nucleic acid sequence andforms a 5′ flap. According to this embodiment, a cleavage structurecomprises a 5′ flap formed by a non-complementary region of theoligonucleotide.

A cleavage structure according to the invention also comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of annealing to a target nucleic acid sequence (for example A inFIG. 3) is complementary to 1 (or more) base pair of the downstreamoligonucleotide (for example C in FIG. 3) that is annealed to a targetnucleic acid sequence and wherein the 1 (or more) base pair overlap isdirectly downstream of the point of extension of the single strandedflap and is formed according to method described in the section entitled“Cleavage Structure”.

II. Nucleic Acid Polymerases

The invention provides for nucleic acid polymerases. Preferably, thenucleic acid polymerase according to the invention is thermostable.

Known DNA polymerases include, for example, E. coli DNA polymerase I,Thermus thermophilus (Tth) DNA polymerase, Bacillus stearothermophilusDNA polymerase, Thermococcus litoralis DNA polymerase, Thermus aquaticus(Taq) DNA polymerase and Pyrococcus furiosus (Pfu) DNA polymerase.

Nucleic acid polymerases substantially lacking 5′ to 3′ exonucleaseactivity useful according to the invention include but are not limitedto Klenow and Klenow exo-, and T7 DNA polymerase (Sequenase).

Thermostable nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity useful according to the invention include but arenot limited to Pfu, exo-Pfu (a mutant form of Pfu that lacks 3′ to 5′exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst,N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form ofVent that lacks 3′ to 5′ exonuclease activity), Deep Vent, Deep Ventexo- (a mutant form of Deep Vent that lacks 3′ to 5′ exonucleaseactivity), U1Tma, and ThermoSequenase.

Nucleic acid polymerases useful according to the invention include bothnative polymerases as well as polymerase mutants, which lack 5′ to 3′exonuclease activity. Nucleic acid polymerases useful according to theinvention can possess different degrees of thermostability. Preferably,a nucleic acid polymerase according to the invention exhibits stranddisplacement activity at the temperature at which it can extend anucleic acid primer. In a preferred embodiment of the invention, anucleic acid polymerase lacks both 5′ to 3′ and 3′ to 5′ exonucleaseactivity.

Additional nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity with different degrees of thermostability usefulaccording to the invention are listed below.

A. Bacteriophage DNA Polymerases (Useful for 37° C. Assays):

Bacteriophage DNA polymerases are devoid of 5′ to 3′ exonucleaseactivity, as this activity is encoded by a separate polypeptide.Examples of suitable DNA polymerases are T4, T7, and φ29 DNA polymerase.The enzymes available commercially are: T4 (available from many sourcese.g., Epicentre) and T7 (available from many sources, e.g. Epicentre forunmodified and USB for 3′ to 5′ exo⁻ T7 “Sequenase” DNA polymerase).

B. Archaeal DNA Polymerases:

There are 2 different classes of DNA polymerases which have beenidentified in archaea: 1. Family B/pol α type (homologs of Pfu fromPyrococcus furiosus) and 2. pol II type (homologs of P. furiosus DP1/DP22-subunit polymerase). DNA polymerases from both classes have been shownto naturally lack an associated 5′ to 3′ exonuclease activity and topossess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNApolymerases (pol α or pol II) can be derived from archaea with optimalgrowth temperatures that are similar to the desired assay temperatures.Examples of suitable archaea include, but are not limited to:

1. Thermolabile (useful for 37° C. assays)—e.g., Methanococcus voltae

2. Thermostable (useful for non-PCR assays)—e.g., Sulfolobussolfataricus, Sulfolobus acidocaldarium, Methanococcus jannaschi,Thermoplasma acidophilum. It is estimated that suitable archaea exhibitmaximal growth temperatures of ≦80-85° C. or optimal growth temperaturesof ≦70-80° C.

3. Thermostable (useful for PCR assays)—e.g., Pyrococcus species(furiosus, species GB-D, species strain KOD1, woesii, abysii,horikoshii), Thermococcus species (litoralis, species 9° North-7,species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobusfulgidus. It is estimated that suitable archaea would exhibit maximalgrowth temperatures of ≧80-85° C. or optimal growth temperatures of≧70-80° C. Appropriate PCR enzymes from the archaeal pol a DNApolymerase group are commercially available, including KOD (Toyobo), Pfx(Life Technologies, Inc.), Vent (New England BioLabs), Deep Vent (NewEngland BioLabs), and Pwo (Boehringer-Mannheim).

Additional archaea related to those listed above are described in thefollowing references: Archaea: A Laboratory Manual (Robb, F. T. andPlace, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1995 and Thermophilic Bacteria (Kristjansson, J. K., ed.)CRC Press, Inc., Boca Raton, Fla., 1992.

C. Eubacterial DNA Polymerases:

There are 3 classes of eubacterial DNA polymerases, pol I, II, and III.Enzymes in the Pol I DNA polymerase family possess 5′ to 3′ exonucleaseactivity, and certain members also exhibit 3′ to 5′ exonucleaseactivity. Pol II DNA polymerases naturally lack 5′ to 3′ exonucleaseactivity, but do exhibit 3′ to 5′ exonuclease activity. Pol III DNApolymerases represent the major replicative DNA polymerase of the celland are composed of multiple subunits. The pol III catalytic subunitlacks 5′ to 3′ exonuclease activity, but in some cases 3′ to 5′exonuclease activity is located in the same polypeptide.

There are no commercial sources of eubacterial pol II and pol III DNApolymerases.

There are a variety of commercially available Pol I DNA polymerases,some of which have been modified to reduce or abolish 5′ to 3′exonuclease activity. Methods used to eliminate 5′ to 3′ exonucleaseactivity of pol I DNA polymerases include:

-   -   mutagenesis (as described in Xu et al., 1997, J. Mol. Biol.,        268:284 and Kim et al., 1997, Mol. Cells, 7:468).    -   N-truncation by proteolytic digestion (as described in Klenow et        al., 1971, Eur. J. Biochem., 22: 371), or    -   N-truncation by cloning and expressing as C-terminal fragments        (as described in Lawyer et al., 1993, PCR Methods Appl., 2:275).

As for archaeal sources, the assay-temperature requirements determinewhich eubacteria should be used as a source of a DNA polymerase usefulaccording to the invention (e.g., mesophiles, thermophiles,hyperthermophiles).

1. Mesophilic/Thermolabile (Useful for 370 C Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: pol II or the pol III catalytic subunit        from mesophilic eubacteria, such as Escherchia coli,        Streptococcus pneumoniae, Haemophilus influenza, Mycobacterium        species (tuberculosis, leprae)    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Pol I DNA polymerases for N-truncation or        mutagenesis can be isolated from the mesophilic eubacteria        listed above (Ci). A commercially-available eubacterial DNA        polymerase pol I fragment is the Klenow fragment (N-truncated E.        coli pol I; Stratagene).

2. Thermostable (Useful for Non PCR Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or the pol III catalytic subunit        from thermophilic eubacteria, such as Bacillus species (e.g.,        stearothermophilus, caldotenax, caldovelox)    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from thermophilic        eubacteria such as the Bacillus species listed above.        Thermostable N-truncated fragments of B. stearothermophilus DNA        polymerase pol I are commercially available and sold under the        trade names Bst DNA polymerase I large fragment (Bio-Rad and        Isotherm DNA polymerase (Epicentre)). A C-terminal fragment of        Bacillus caldotenax pol I is available from Panvera (sold under        the tradename Ladderman).

3. Thermostable (Useful for PCR Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or pol III catalytic subunit from        Thermus species (aquaticus, thermophilus, flavus, ruber,        caldophilus, filiformis, brokianus) or from Thermotoga maritima.        The catalytic pol III subunits from Thermus thermophilus and        Thermus aquaticus are described in Yi-Ping et al., 1999, J. Mol.        Evol., 48:756 and McHenry et al., 1997, J. Mol. Biol., 272:178.    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from a variety of        thermophilic eubacteria, including Thermus species and        Thermotoga maritima (see above). Thermostable fragments of        Thermus aquaticus DNA polymerase pol I (Taq) are commercially        available and sold under the trade names KlenTaq1 (Ab Peptides),        Stoffel fragment (Perkin-Elmer), and ThermoSequenase (Amersham).        In addition to C-terminal fragments, 5′ to 3′ exonuclease⁻ Taq        mutants are also commercially available, such as TaqFS        (Hoffman-LaRoche). In addition to 5′-3′ exonuclease⁻ versions of        Taq, an N-truncated version of Thermotoga maritima DNA        polymerase I is also commercially available (tradename U1Tma,        Perkin-Elmer).

Additional eubacteria related to those listed above are described inThermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., BocaRaton, Fla., 1992.

D. Eukaryotic 5′ to 3′ Exonuclease⁻ DNA Polymerases (Useful for 370 CAssays)

There are several DNA polymerases that have been identified ineukaryotes, including DNA pol a (replication/repair), δ (replication), ε(replication), β (repair) and γ (mitochondrial replication). EukaryoticDNA polymerases are devoid of 5′ to 3′ exonuclease activity, as thisactivity is encoded by a separate polypeptide (e.g., mammalian FEN-1 oryeast RAD2). Suitable thermolabile DNA polymerases may be isolated froma variety of eukaryotes (including but not limited to yeast, mammaliancells, insect cells, Drosophila) and eukaryotic viruses (e.g., EBV,adenovirus).

It is possible that DNA polymerase mutants lacking 3′-5′ exonuclease(proofreading) activity, in addition to lacking 5′ to 3′ exonucleaseactivity, could exhibit improved performance in FEN-based detectionstrategies. For example, reducing or abolishing inherent 3′ to 5′exonuclease activity may lower background signals by diminishingnon-specific exonucleolytic degradation of labeled probes. Three 3′ to5′ exonuclease motifs have been identified, and mutations in theseregions have been shown to abolish 3′ to 5′ exonuclease activity inKlenow, φ29, T4, T7, and Vent DNA polymerases, yeast Pol α, Pol β, andPol γ, and Bacillus subtilis Pol III (reviewed in Derbeyshire et al.,1995, Methods. Enzymol. 262:363). Methods for preparing additional DNApolymerase mutants, with reduced or abolished 3′ to 5′ exonucleaseactivity, are well known in the art.

Commercially-available enzymes that lack both 5′ to 3′ and 3′ to 5′exonuclease activities include Sequenase (exo⁻ T7; USB), Pfu exo⁻(Stratagene), exo⁻ Vent (New England BioLabs), exo⁻ DeepVent (NewEngland BioLabs), exo⁻ Klenow fragment (Stratagene), Bst (Bio-Rad),Isotherm (Epicentre), Ladderman (Panvera), KlenTaq1 (Ab Peptides),Stoffel fragment (Perkin-Elmer), ThermoSequenase (USB), and TaqFS(Hoffman-LaRoche).

If polymerases other than Pfu are used, buffers and extensiontemperatures are selected to allow for optimal activity by theparticular polymerase useful according to the invention. Buffers andextension temperatures useful for polymerases according to the inventionare know in the art and can also be determined from the Vendor'sspecifications.

III. Nucleic Acids

A. Nucleic Acid Sequences Useful in the Invention

The invention provides for methods of detecting or measuring a targetnucleic acid sequence; and also utilizes oligonucleotides, primers andprobes for forming a cleavage structure according to the invention andprimers for amplifying a template nucleic acid sequence. As used herein,the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” referto primers, probes, and oligomer fragments to be detected, and shall begeneric to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases (including abasic sites). Thereis no intended distinction in length between the term “nucleic acid”,“polynucleotide” and “oligonucleotide”, and these terms will be usedinterchangeably. These terms refer only to the primary structure of themolecule. Thus, these terms include double- and single-stranded DNA, aswell as double- and single-stranded RNA.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.”

The oligonucleotide is not necessarily physically derived from anyexisting or natural sequence but may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription ora combination thereof. The terms “oligonucleotide” or “nucleic acid”intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, orsynthetic origin which, by virtue of its synthetic origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points toward the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide.

Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids of the present invention and include, forexample, inosine and 7-deazaguanine. Complementarity need not beperfect; stable duplexes may contain mismatched base pairs or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which half of the basepairs have disassociated.

B. Primers and Probes Useful According to the Invention

The invention provides for oligonucleotide primers and probes useful fordetecting or measuring a nucleic acid, for amplifying a template nucleicacid sequence, and for forming a cleavage structure according to theinvention.

The term “primer” may refer to more than one primer and refers to anoligonucleotide, whether occurring naturally, as in a purifiedrestriction digest, or produced synthetically, which is capable ofacting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand iscatalyzed. Such conditions include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as DNA polymerase or reverse transcriptase, in a suitable buffer(“buffer” includes substituents which are cofactors, or which affect pH,ionic strength, etc.), and at a suitable temperature. The primer ispreferably single-stranded for maximum efficiency in amplification.

Oligonucleotide primers useful according to the invention aresingle-stranded DNA or RNA molecules that are hybridizable to a templatenucleic acid sequence and prime enzymatic synthesis of a second nucleicacid strand. The primer is complementary to a portion of a targetmolecule present in a pool of nucleic acid molecules. It is contemplatedthat oligonucleotide primers according to the invention are prepared bysynthetic methods, either chemical or enzymatic. Alternatively, such amolecule or a fragment thereof is naturally-occurring, and is isolatedfrom its natural source or purchased from a commercial supplier.Oligonucleotide primers and probes are 5 to 100 nucleotides in length,ideally from 17 to 40 nucleotides, although primers and probes ofdifferent length are of use. Primers for amplification are preferablyabout 17-25 nucleotides. Primers for the production of a cleavagestructure according to the invention are preferably about 17-45nucleotides. Primers useful according to the invention are also designedto have a particular melting temperature (Tm) by the method of meltingtemperature estimation. Commercial programs, including Oligo™, PrimerDesign and programs available on the internet, including Primer3 andOligo Calculator can be used to calculate a Tm of a nucleic acidsequence useful according to the invention. Preferably, the Tm of anamplification primer useful according to the invention, as calculatedfor example by Oligo Calculator, is preferably between about 45 and 65°C. and more preferably between about 50 and 60° C. Preferably, the Tm ofa probe useful according to the invention is 7° C. higher than the Tm ofthe corresponding amplification primers.

As used herein, “probe” refers to a labeled primer useful forpreparation of a cleavage structure according to the invention. Pairs ofsingle-stranded DNA primers can be annealed to sequences within a targetnucleic acid sequence or can be used to prime amplifying DNA synthesisof a target nucleic acid sequence.

Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203,incorporated herein by reference. As a result, it is expected that acertain degree of mismatch at the priming site is tolerated. Suchmismatch may be small, such as a mono-, di- or tri-nucleotide.Alternatively, a region of mismatch may encompass loops, which aredefined as regions in which there exists a mismatch in an uninterruptedseries of four or more nucleotides.

Numerous factors influence the efficiency and selectivity ofhybridization of the primer to a second nucleic acid molecule. Thesefactors, which include primer length, nucleotide sequence and/orcomposition, hybridization temperature, buffer composition and potentialfor steric hindrance in the region to which the primer is required tohybridize, will be considered when designing oligonucleotide primersaccording to the invention.

A positive correlation exists between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence. In particular, longer sequences have a higher meltingtemperature (T_(M)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization. Primer sequences with a high G-C content or that comprisepalindromic sequences tend to self-hybridize, as do their intendedtarget sites, since unimolecular, rather than bimolecular, hybridizationkinetics are generally favored in solution. However, it is alsoimportant to design a primer that contains sufficient numbers of G-Cnucleotide pairings since each G-C pair is bound by three hydrogenbonds, rather than the two that are found when A and T bases pair tobind the target sequence, and therefore forms a tighter, stronger bond.Hybridization temperature varies inversely with primer annealingefficiency, as does the concentration of organic solvents, e.g.formamide, that might be included in a priming reaction or hybridizationmixture, while increases in salt concentration facilitate binding. Understringent annealing conditions, longer hybridization probes, orsynthesis primers, hybridize more efficiently than do shorter ones,which are sufficient under more permissive conditions. Stringenthybridization conditions typically include salt concentrations of lessthan about 1M, more usually less than about 500 mM and preferably lessthan about 200 mM. Hybridization temperatures range from as low as 0° C.to greater than 22° C., greater than about 30° C., and (most often) inexcess of about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As several factors affect thestringency of hybridization, the combination of parameters is moreimportant than the absolute measure of a single factor.

Oligonucleotide primers can be designed with these considerations inmind and synthesized according to the following methods.

1. Oligonucleotide Primer Design Strategy

The design of a particular oligonucleotide primer for the purpose ofsequencing, PCR or for the preparation of a cleavage structure accordingto the invention, involves selecting a sequence that is capable ofrecognizing the target sequence, but has a minimal predicted secondarystructure. The oligonucleotide sequence binds only to a single site inthe target nucleic acid sequence. Furthermore, the Tm of theoligonucleotide is optimized by analysis of the length and GC content ofthe oligonucleotide. Furthermore, when designing a PCR primer useful forthe amplification of genomic DNA, the selected primer sequence does notdemonstrate significant matches to sequences in the GenBank database (orother available databases).

The design of a primer is facilitated by the use of readily availablecomputer programs, developed to assist in the evaluation of the severalparameters described above and the optimization of primer sequences.Examples of such programs are “PrimerSelect” of the DNAStar™ softwarepackage (DNAStar, Inc.; Madison, Wis.), OLIGO 4.0 (National Biosciences,Inc.), PRIMER, Oligonucleotide Selection Program, PGEN and Amplify(described in Ausubel et al., 1995, Short Protocols in MolecularBiology, 3rd Edition, John Wiley & Sons). In one embodiment, primers aredesigned with sequences that serve as targets for other primers toproduce a PCR product that has known sequences on the ends which serveas targets for further amplification (e.g. to sequence the PCR product).If many different target nucleic acid sequences are amplified withspecific primers that share a common ‘tail’ sequence’, the PCR productsfrom these distinct genes can subsequently be sequenced with a singleset of primers. Alternatively, in order to facilitate subsequent cloningof amplified sequences, primers are designed with restriction enzymesite sequences appended to their 5′ ends. Thus, all nucleotides of theprimers are derived from a target nucleic acid sequence or sequencesadjacent to a target nucleic acid sequence, except for the fewnucleotides necessary to form a restriction enzyme site. Such enzymesand sites are well known in the art. If the genomic sequence of a targetnucleic acid sequence and the sequence of the open reading frame of atarget nucleic acid sequence are known, design of particular primers iswell within the skill of the art.

It is well known by those with skill in the art that oligonucleotidescan be synthesized with certain chemical and/or capture moieties, suchthat they can be coupled to solid supports. Suitable capture moietiesinclude, but are not limited to, biotin, a hapten, a protein, anucleotide sequence, or a chemically reactive moiety. Sucholigonucleotides may either be used first in solution, and then capturedonto a solid support, or first attached to a solid support and then usedin a detection reaction. An example of the latter would be to couple adownstream probe molecule to a solid support, such that the 5′ end ofthe downstream probe molecule comprised a fluorescent quencher. The samedownstream probe molecule would also comprise a fluorophore in alocation such that a FEN nuclease cleavage would physically separate thequencher from the fluorophore. For example, the target nucleic acidcould hybridize with the solid-phase downstream probe oligonucleotide,and a liquid phase upstream primer could also hybridize with the targetmolecule, such that a FEN cleavage reaction occurs on the solid supportand liberates the 5′ quencher moiety from the complex. This would causethe solid support-bound fluorophore to be detectable, and thus revealthe presence of a cleavage event upon a suitably labeled or identifiedsolid support. Different downstream probe molecules could be bound todifferent locations on an array. The location on the array wouldidentify the probe molecule, and indicate the presence of the templateto which the probe molecule can hybridize.

2. Synthesis

The primers themselves are synthesized using techniques that are alsowell known in the art. Methods for preparing oligonucleotides ofspecific sequence are known in the art, and include, for example,cloning and restriction digest analysis of appropriate sequences anddirect chemical synthesis. Once designed, oligonucleotides are preparedby a suitable chemical synthesis method, including, for example, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology, 68:90, the phosphodiester method disclosed by Brown et al.,1979, Methods in Enzymology, 68:109, the diethylphosphoramidate methoddisclosed in Beaucage et al., 1981, Tetrahedron Letters, 22:1859, andthe solid support method disclosed in U.S. Pat. No. 4,458,066, or byother chemical methods using either a commercial automatedoligonucleotide synthesizer (which is commercially available) or VLSIPS™technology.

C. Probes

The invention provides for probes useful for forming a labeled cleavagestructure as defined herein. Methods of preparing a labeled cleavagestructure according to the invention are provided in the sectionentitled “Cleavage Structure” below.

As used herein, the term “probe” refers to a labeled oligonucleotidewhich forms a duplex structure with a sequence in the target nucleicacid, due to complementarity of at least one sequence in the probe witha sequence in the target region. The probe, preferably, does not containa sequence complementary to sequence(s) used in the primer extension(s). Generally the 3′ terminus of the probe will be “blocked” toprohibit incorporation of the probe into a primer extension product.“Blocking” can be achieved by using non-complementary bases or by addinga chemical moiety such as biotin or a phosphate group to the 3′ hydroxlof the last nucleotide, which may, depending upon the selected moiety,serve a dual purpose by also acting as a label for subsequent detectionor capture of the nucleic acid attached to the label. Blocking can alsobe achieved by removing the 3′-OH or by using a nucleotide that lacks a3′-OH such as dideoxynucleotide.

Methods of labeling a probe according to the invention and suitablelabels are described below in the section entitled “Cleavage Structure”.

D. Production of a Nucleic Acid

The invention provides nucleic acids to be detected and or measured, foramplification of a target nucleic acid sequence and for formation of acleavage structure.

The present invention utilizes nucleic acids comprising RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers. The invention includesboth sense and antisense strands of a nucleic acid. According to theinvention, the nucleic acid may be chemically or biochemically modifiedor may contain non-natural or derivatized nucleotide bases. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g. methylphosphonates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators, (e.g. acridine, psoralen, etc.) chelators,alkylators, and modified linkages (e.g. alpha anomeric nucleic acids,etc.) Also included are synthetic molecules that mimic polynucleotidesin their ability to bind to a designated sequence via hydrogen bondingand other chemical interactions. Such molecules are known in the art andinclude, for example, those in which peptide linkages substitute forphosphate linkages in the backbone of the molecule.

1. Nucleic Acids Comprising DNA

a. Cloning

Nucleic acids comprising DNA can be isolated from cDNA or genomiclibraries by cloning methods well known to those skilled in the art(Ausubel et al., supra). Briefly, isolation of a DNA clone comprising aparticular nucleic acid sequence involves screening a recombinant DNA orcDNA library and identifying the clone containing the desired sequence.Cloning will involve the following steps. The clones of a particularlibrary are spread onto plates, transferred to an appropriate substratefor screening, denatured, and probed for the presence of a particularnucleic acid. A description of hybridization conditions, and methods forproducing labeled probes is included below.

The desired clone is preferably identified by hybridization to a nucleicacid probe or by expression of a protein that can be detected by anantibody. Alternatively, the desired clone is identified by polymerasechain amplification of a sequence defined by a particular set of primersaccording to the methods described below.

The selection of an appropriate library involves identifying tissues orcell lines that are an abundant source of the desired sequence.Furthermore, if a nucleic acid of interest contains regulatory sequenceor intronic sequence a genomic library is screened (Ausubel et al.,supra).

b. Genomic DNA

Nucleic acid sequences of the invention are amplified from genomic DNA.Genomic DNA is isolated from tissues or cells according to the followingmethod.

To facilitate detection of a variant form of a gene from a particulartissue, the tissue is isolated free from surrounding normal tissues. Toisolate genomic DNA from mammalian tissue, the tissue is minced andfrozen in liquid nitrogen. Frozen tissue is ground into a fine powderwith a prechilled mortar and pestle, and suspended in digestion buffer(100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH 8.0, 0.5% (w/v)SDS, 0.1 mg/ml proteinase K) at 1.2 ml digestion buffer per 100 mg oftissue. To isolate genomic DNA from mammalian tissue culture cells,cells are pelleted by centrifugation for 5 min at 500×g, resuspended in1-10 ml ice-cold PBS, repelleted for 5 min at 500×g and resuspended in 1volume of digestion buffer.

Samples in digestion buffer are incubated (with shaking) for 12-18 hoursat 50° C., and then extracted with an equal volume ofphenol/chloroform/isoamyl alcohol. If the phases are not resolvedfollowing a centrifugation step (10 min at 1700×g), another volume ofdigestion buffer (without proteinase K) is added and the centrifugationstep is repeated. If a thick white material is evident at the interfaceof the two phases, the organic extraction step is repeated. Followingextraction the upper, aqueous layer is transferred to a new tube towhich will be added 1/2 volume of 7.5M ammonium acetate and 2 volumes of100% ethanol. The nucleic acid is pelleted by centrifugation for 2 minat 1700×g, washed with 70% ethanol, air dried and resuspended in TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) at 1 mg/ml. ResidualRNA is removed by incubating the sample for 1 hour at 370 C in thepresence of 0.1% SDS and 1 μg/ml DNase-free RNase, and repeating theextraction and ethanol precipitation steps. The yield of genomic DNA,according to this method is expected to be approximately 2 mg DNA/1 gcells or tissue (Ausubel et al., supra). Genomic DNA isolated accordingto this method can be used for PCR analysis, according to the invention.

c. Restriction Digest (of cDNA or Genomic DNA)

Following the identification of a desired cDNA or genomic clonecontaining a particular target nucleic acid sequence, nucleic acids ofthe invention may be isolated from these clones by digestion withrestriction enzymes.

The technique of restriction enzyme digestion is well known to thoseskilled in the art (Ausubel et al., supra). Reagents useful forrestriction enzyme digestion are readily available from commercialvendors including Stratagene, as well as other sources.

d. PCR

Nucleic acids of the invention may be amplified from genomic DNA orother natural sources by the polymerase chain reaction (PCR). PCRmethods are well-known to those skilled in the art.

PCR provides a method for rapidly amplifying a particular DNA sequenceby using multiple cycles of DNA replication catalyzed by a thermostable,DNA-dependent DNA polymerase to amplify the target sequence of interest.PCR requires the presence of a target nucleic acid sequence to beamplified, two single stranded oligonucleotide primers flanking thesequence to be amplified, a DNA polymerase, deoxyribonucleosidetriphosphates, a buffer and salts.

PCR, is performed as described in Mullis and Faloona, 1987, MethodsEnzymol., 155: 335, herein incorporated by reference.

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science230:1350.

PCR is performed using template DNA (at least 1 fg; more usefully,1-1000 ng) and at least 25 pmol of oligonucleotide primers. A typicalreaction mixture includes: 2 μl of DNA, 25 pmol of oligonucleotideprimer, 2.5 μl of a suitable buffer, 0.4 μl of 1.25 μM dNTP, 2.5 unitsof Taq DNA polymerase (Stratagene) and deionized water to a total volumeof 25 μl. Mineral oil is overlaid and the PCR is performed using aprogrammable thermal cycler.

The length and temperature of each step of a PCR cycle, as well as thenumber of cycles, are adjusted according to the stringency requirementsin effect. Annealing temperature and timing are determined both by theefficiency with which a primer is expected to anneal to a template andthe degree of mismatch that is to be tolerated. The ability to optimizethe stringency of primer annealing conditions is well within theknowledge of one of moderate skill in the art. An annealing temperatureof between 30° C. and 72° C. is used. Initial denaturation of thetemplate molecules normally occurs at between 92° C. and 99° C. for 4minutes, followed by 20-40 cycles consisting of denaturation (94-99° C.for 15 seconds to 1 minute), annealing (temperature determined asdiscussed above; 1-2 minutes), and extension (72° C. for 1 minute). Thefinal extension step is generally carried out for 4 minutes at 72° C.,and may be followed by an indefinite (0-24 hour) step at 4° C.

Detection methods generally employed in standard PCR techniques use alabeled probe with the amplified DNA in a hybridization assay.Preferably, the probe is labeled, e.g., with ³²P, biotin, horseradishperoxidase (HRP), etc., to allow for detection of hybridization.

Other means of detection include the use of fragment length polymorphism(PCR FLP), hybridization to allele-specific oligonucleotide (ASO) probes(Saiki et al., 1986, Nature 324:163), or direct sequencing via thedideoxy method (using amplified DNA rather than cloned DNA). Thestandard PCR technique operates (essentially) by replicating a DNAsequence positioned between two primers, providing as the major productof the reaction a DNA sequence of discrete length terminating with theprimer at the 5′ end of each strand. Thus, insertions and deletionsbetween the primers result in product sequences of different lengths,which can be detected by sizing the product in PCR-FLP. In an example ofASO hybridization, the amplified DNA is fixed to a nylon filter (by, forexample, UV irradiation) in a series of “dot blots”, then allowed tohybridize with an oligonucleotide probe labeled with HRP under stringentconditions. After washing, terramethylbenzidine (TMB) and hydrogenperoxide are added: HRP oxidizes the hydrogen peroxide, which in turnoxidizes the TMB to a blue precipitate, indicating a hybridized probe.

A PCR assay for detecting or measuring a nucleic assay according to theinvention is described in the section entitled “Methods of Use”.

2. Nucleic Acids Comprising RNA

The present invention also provides a nucleic acid comprising RNA.

Nucleic acids comprising RNA can be purified according to methods wellknown in the art (Ausubel et al., supra). Total RNA can be isolated fromcells and tissues according to methods well known in the art (Ausubel etal., supra) and described below.

RNA is purified from mammalian tissue according to the following method.Following removal of the tissue of interest, pieces of tissue of ≦2 gare cut and quick frozen in liquid nitrogen, to prevent degradation ofRNA. Upon the addition of a suitable volume of guanidinium solution (forexample 20 ml guanidinium solution per 2 g of tissue), tissue samplesare ground in a tissuemizer with two or three 10-second bursts. Toprepare tissue guanidinium solution (1 L) 590.8 g guanidiniumisothiocyanate is dissolved in approximately 400 ml DEPC-treated H₂O. 25ml of 2 M Tris-HCl, pH 7.5 (0.05 M final) and 20 ml Na₂EDTA (0.01 Mfinal) is added, the solution is stirred overnight, the volume isadjusted to 950 ml, and 50 ml 2-ME is added.

Homogenized tissue samples are subjected to centrifugation for 10 min at12,000×g at 12° C. The resulting supernatant is incubated for 2 min at65° C. in the presence of 0.1 volume of 20% Sarkosyl, layered over 9 mlof a 5.7M CsCl solution (0.1 g CsCl/ml), and separated by centrifugationovernight at 113,000×g at 22° C. After careful removal of thesupernatant, the tube is inverted and drained. The bottom of the tube(containing the RNA pellet) is placed in a 50 ml plastic tube andincubated overnight (or longer) at 40 C in the presence of 3 ml tissueresuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) toallow complete resuspension of the RNA pellet. The resulting RNAsolution is extracted sequentially with 25:24:1phenol/chloroform/isoamyl alcohol, followed by 24:1 chloroform/isoamylalcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin etal., 1979, Biochemistry, 18: 5294).

Alternatively, RNA is isolated from mammalian tissue according to thefollowing single step protocol. The tissue of interest is prepared byhomogenization in a glass teflon homogenizer in 1 ml denaturing solution(4M guanidinium thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M 2-ME,0.5% (w/v) N-laurylsarkosine) per 100 mg tissue. Following transfer ofthe homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodiumacetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1chloroform/isoamyl alcohol are added sequentially. The sample is mixedafter the addition of each component, and incubated for 15 min at 0-4°C. after all components have been added. The sample is separated bycentrifugation for 20 min at 10,000×g, 4° C., precipitated by theaddition of 1 ml of 100% isopropanol, incubated for 30 minutes at −20°C. and pelleted by centrifugation for 10 minutes at 10,000×g, 4° C. Theresulting RNA pellet is dissolved in 0.3 ml denaturing solution,transferred to a microfuge tube, precipitated by the addition of 0.3 mlof 100% isopropanol for 30 minutes at −20° C., and centrifuged for 10minutes at 10,000×g at 4° C. The RNA pellet is washed in 70% ethanol,dried, and resuspended in 100-200

l DEPC-treated water or DEPC-treated 0.5% SDS (Chomczynski and Sacchi,1987, Anal. Biochem., 162: 156).

Nucleic acids comprising RNA can be produced according to the method ofin vitro transcription.

The technique of in vitro transcription is well known to those of skillin the art. Briefly, the gene of interest is inserted into a vectorcontaining an SP6, T3 or T7 promoter. The vector is linearized with anappropriate restriction enzyme that digests the vector at a single sitelocated downstream of the coding sequence. Following a phenol/chloroformextraction, the DNA is ethanol precipitated, washed in 70% ethanol,dried and resuspended in sterile water. The in vitro transcriptionreaction is performed by incubating the linearized DNA withtranscription buffer (200 mM Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mMspermidine, 250 NaCl [T7 or T3] or 200 mM Tris-HCl, pH 7.5, 30 mM MgCl₂,10 mM spermidine [SP6]), dithiothreitol, RNase inhibitors, each of thefour ribonucleoside triphosphates, and either SP6, T7 or T3 RNApolymerase for 30 min at 37° C. To prepare a radiolabeled polynucleotidecomprising RNA, unlabeled UTP will be omitted and ³⁵S-UTP will beincluded in the reaction mixture. The DNA template is then removed byincubation with DNaseI. Following ethanol precipitation, an aliquot ofthe radiolabeled RNA is counted in a scintillation counter to determinethe cpm/μl (Ausubel et al., supra).

Alternatively, nucleic acids comprising RNA are prepared by chemicalsynthesis techniques such as solid phase phosphoramidite (describedabove).

3. Nucleic Acids Comprising Oligonucleotides

A nucleic acid comprising oligonucleotides can be made by usingoligonucleotide synthesizing machines which are commercially available(described above).

IV. Cleavage Structure

The invention provides for a cleavage structure that can be cleaved by aFEN nuclease, and therefore teaches methods of preparing a cleavagestructure. The invention also provides a labeled cleavage structure andmethods of preparing a labeled cleavage structure.

A. Preparation of a Cleavage Structure

A cleavage structure according to the invention is formed by incubatinga) an upstream, preferably extendable 3′ end, preferably anoligonucleotide primer, b) an oligonucleotide probe located not morethan 5000 nucleotides downstream of the upstream primer and c) anappropriate target nucleic acid sequence wherein the target sequence iscomplementary to both primers and d) a suitable buffer (for exampleSentinel Molecular Beacon PCR core buffer (Catalog #600500) or 10×Pfubuffer available from Stratagene (Catalog #200536), under conditionsthat allow the nucleic acid sequence to hybridize to the oligonucleotideprimers (for example 95° C. for 2-5 minutes followed by cooling tobetween approximately 50-60° C.). The optimal temperature will varydepending on the specific probe(s), primers and polymerases. Inpreferred embodiments of the invention a cleavage structure comprises anoverlapping flap wherein the 3′ end of an upstream oligonucleotidecapable of hybridizing to a target nucleic acid sequence (for example Ain FIG. 3) is complementary to 1 or more base pair(s) of the downstreamoligonucleotide (for example C in FIG. 3) that is annealed to a targetnucleic acid sequence and wherein the 1 base pair overlap is directlydownstream of the point of extension of the single stranded flap.

The 3′ end of the upstream oligonucleotide primer is extended by thesynthetic activity of a polymerase according to the invention such thatthe newly synthesized 3′ end of the upstream oligonucleotide primerpartially displaces the 5′ end of the downstream oligonucleotide probe.Extension is preferably carried out in the presence of 1× SentinelMolecular beacon core buffer or 1×Pfu buffer for 15 seconds at 72° C. Inone embodiment of the invention, a cleavage structure according to theinvention can be prepared by incubating a target nucleic acid sequencewith a partially complementary oligonucleotide primer such that the 3′complementary region anneals to the target nucleic acid sequence and thenon-complementary 5′ region that does not anneal to the target nucleicacid sequence forms a 5′ flap. Annealing is preferably carried out underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primer (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.) in the presence a suitablebuffer (for example 1× Sentinel Molecular beacon core buffer or 1×Pfubuffer.

B. How to Prepare a Labeled Cleavage Structure

In the present invention, a label is attached to an oligonucleotideprimer comprising the cleavage structure, thereby forming a probe. Thus,the cleaved mononucleotides or small oligonucleotides which are cleavedby the endonuclease activity of the flap-specific nuclease can bedetected.

A labeled cleavage structure according to the invention is formed byincubating a) an upstream extendable 3′ end, preferably anoligonucleotide primer, b) a labeled probe located not more than 500nucleotides downstream of the upstream primer and c) an appropriatetarget nucleic acid sequence wherein the target sequence iscomplementary to the oligonucleotides and d) a suitable buffer (forexample 1× Sentinel Molecular beacon core buffer or 1×Pfu buffer), underconditions that allow the nucleic acid sequence to hybridize to theoligonucleotide primers (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.). A cleavage structureaccording to the invention also comprises an overlapping flap whereinthe 3′ end of an upstream oligonucleotide capable of hybridizing to atarget nucleic acid sequence (for example A in FIG. 3) is complementaryto 1 base pair of the downstream oligonucleotide (for example C in FIG.3) that is annealed to a target nucleic acid sequence and wherein the 1base pair overlap is directly downstream of the point of extension ofthe single stranded flap. The 3′ end of the upstream primer is extendedby the synthetic activity of a polymerase such that the newlysynthesized 3′ end of the upstream primer partially displaces thelabeled 5′ end of the downstream probe. Extension is preferably carriedout in the presence of 1× Sentinel Molecular beacon core buffer or 1×Pfubuffer for 15 seconds at 72° C. A cleavage structure according to theinvention can be prepared by incubating a target nucleic acid sequencewith a probe comprising a non-complementary, labeled, 5′ region thatdoes not anneal to the target nucleic acid sequence and forms a 5′ flap,and a complementary 3′ region that anneals to the target nucleic acidsequence. Annealing is preferably carried out under conditions thatallow the nucleic acid sequence to hybridize to the oligonucleotideprimer (for example 95° C. for 2-5 minutes followed by cooling tobetween approximately 50-60° C.) in the presence a suitable buffer (forexample 1× Sentinel Molecular beacon core buffer or 1×Pfu buffer).

Subsequently, any of several strategies may be employed to distinguishthe uncleaved labeled nucleic acid from the cleaved fragments thereof.In this manner, the present invention permits identification of thosesamples that contain a target nucleic acid sequence.

The oligonucleotide probe is labeled, as described below, byincorporating moieties detectable by spectroscopic, photochemical,biochemical, immunochemical, enzymatic or chemical means. The method oflinking or conjugating the label to the oligonucleotide probe depends,of course, on the type of label(s) used and the position of the label onthe probe. Preferably a probe is labeled at the 5′ end although probeslabeled at the 3′ end or labeled throughout the length of the probe arealso useful in particular embodiments of the invention.

A variety of labels that would be appropriate for use in the invention,as well as methods for their inclusion in the probe, are known in theart and include, but are not limited to, enzymes (e.g., alkalinephosphatase and horseradish peroxidase) and enzyme substrates,radioactive atoms, fluorescent dyes, chromophores, chemiluminescentlabels, electrochemiluminescent labels, such as Origen™ (Igen), that mayinteract with each other to enhance, alter, or diminish a signal. Ofcourse, if a labeled molecule is used in a PCR based assay carried outusing a thermal cycler instrument, the label must be able to survive thetemperature cycling required in this automated process.

Among radioactive atoms, ³³P or, ³²P is preferred. Methods forintroducing ³³P or, ³²P into nucleic acids are known in the art, andinclude, for example, 5′ labeling with a kinase, or random insertion bynick translation. “Specific binding partner” refers to a protein capableof binding a ligand molecule with high specificity, as for example inthe case of an antigen and a monoclonal antibody specific therefor.Other specific binding partners include biotin and avidin orstreptavidin, IgG and protein A, and the numerous receptor-ligandcouples known in the art. The above description is not meant tocategorize the various labels into distinct classes, as the same labelmay serve in several different modes. For example, ¹²⁵I may serve as aradioactive label or as an electron-dense reagent. HRP may serve as anenzyme or as antigen for a monoclonal antibody. Further, one may combinevarious labels for desired effect. For example, one might label a probewith biotin, and detect the presence of the probe with avidin labeledwith ¹²⁵I, or with an anti-biotin monoclonal antibody labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art and are considered as equivalents withinthe scope of the instant invention.

Fluorophores for use as labels in constructing labeled probes of theinvention include rhodamine and derivatives (such as Texas Red),fluorescein and derivatives (such as 5-bromomethyl fluorescein), LuciferYellow, IAEDANS, 7-Me₂N-coumarin-4-acetate,7-OH-4-CH₃-coumarin-3-acetate, 7-NH₂-4-CH₃-coumarin-3-acetate (AMCA),monobromobimane, pyrene trisulfonates, such as Cascade Blue, andmonobromorimethyl-ammoniobimane. In general, fluorophores with wideStokes shifts are preferred, to allow using fluorimeters with filtersrather than a monochromometer and to increase the efficiency ofdetection.

Probes labeled with fluorophores can readily be used in FEN mediatedcleavage of a cleavage structure comprising a labeled probe according tothe invention. If the label is on the 5′-end of the probe, the FENgenerated labeled fragment is separated from the intact, hybridizedprobe by procedures well known in the art. The fluorescence of thereleased label is then compared to the label remaining bound to thetarget. It is not necessary to separate the FEN generated fragment andthe probe that remains bound to the target after cleavage in thepresence of FEN if the probe is synthesized with a fluorophore, usuallyat the 5′-end, and a quencher, usually about 20 nucleotides downstreamof the dye. Such a dual labeled probe will not fluoresce when intactbecause the light emitted from the dye is quenched by the quencher.Thus, any fluorescence emitted by an intact probe is considered to bebackground fluorescence. When a labeled probe is cleaved by a FENnuclease, dye and quencher are separated and the released fragment willfluoresce. The amount of fluorescence is proportional to the amount ofnucleic acid target sequence present in a sample.

In some situations, one can use two interactive labels on a singleoligonucleotide with due consideration given for maintaining anappropriate spacing of the labels on the oligonucleotide to permit theseparation of the labels during oligonucleotide hydrolysis. Preferredinteractive labels useful according to the invention include, but arenot limited to rhodamine and derivatives, fluorescein and derivatives,Texas Red, coumarin and derivatives, crystal violet and include, but arenot limited to DABCYL, TAMRA and NTB (nitrothiazole blue).

In another embodiment of the invention, detection of the hydrolyzed,labeled probe can be accomplished using, for example, fluorescencepolarization, a technique to differentiate between large and smallmolecules based on molecular tumbling. Large molecules (i.e., intactlabeled probe) tumble in solution much more slowly than small molecules.Upon linkage of a fluorescent moiety to the molecule of interest (e.g.,the 5′ end of a labeled probe), this fluorescent moiety can be measured(and differentiated) based on molecular tumbling, thus differentiatingbetween intact and digested probe.

In yet another embodiment, two labeled nucleic acids are used, eachcomplementary to separate regions of separate strands of adouble-stranded target sequence, but not to each other, so that alabeled nucleic acid anneals downstream of each primer. For example, thepresence of two probes can potentially double the intensity of thesignal generated from a single label and may further serve to reduceproduct strand reannealing, as often occurs during PCR amplification.The probes are selected so that the probes bind at positions adjacent(downstream) to the positions at which primers bind.

One can also use multiple probes in the present invention to achieveother benefits. For instance, one could test for any number of pathogensin a sample simply by putting as many probes as desired into thereaction mixture; the probes could each comprise a different label tofacilitate detection.

One can also achieve allele-specific or species-specific discriminationusing multiple probes in the present invention, for instance, by usingprobes that have different T_(m)s and conducting the annealing/cleavagereaction at a temperature specific for only one probe/allele duplex. Onecan also achieve allele specific discrimination by using only a singleprobe and examining the types of cleavage products generated. In thisembodiment of the invention, the probe is designed to be exactlycomplementary, at least in the 5′ terminal region, to one allele but notto the other allele(s). With respect to the other allele(s), the probewill be mismatched in the 5′ terminal region of the probe so that adifferent cleavage product will be generated as compared to the cleavageproduct generated when the probe is hybridized to the exactlycomplementary allele.

Although probe sequence can be selected to achieve important benefits,one can also realize important advantages by selection of probelabels(s). The labels may be attached to the oligonucleotide directly orindirectly by a variety of techniques. Depending on the precise type oflabel used, the label can be located at the 5′ or 3′ end of the probe,located internally in the probe, or attached to spacer arms of varioussizes and compositions to facilitate signal interactions. Usingcommercially available phosphoramidite reagents, one can produceoligomers containing functional groups (e.g., thiols or primary amines)at either the 5- or the 3-terminus via an appropriately protectedphosphoramidite, and can label them using protocols described in, forexample, PCR Protocols: A Guide to Methods and Applications, Innis etal., eds. Academic Press, Ind., 1990.

Methods for introducing oligonucleotide functionalizing reagents tointroduce one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide probe sequence, typically at the 5′ terminus, aredescribed in U.S. Pat. No. 4,914,210. A 5′ phosphate group can beintroduced as a radioisotope by using polynucleotide kinase andgamma-³²P-ATP or gamma-³³P-ATP to provide a reporter group. Biotin canbe added to the 5′ end by reacting an aminothymidine residue, or a6-amino hexyl residue, introduced during synthesis, with anN-hydroxysuccinimide ester of biotin. Labels at the 3′ terminus mayemploy polynucleotide terminal transferase to add the desired moiety,such as for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

Oligonucleotide derivatives are also available labels. For example,etheno-dA and etheno-A are known fluorescent adenine nucleotides thatcan be incorporated into a nucleic acid probe. Similarly, etheno-dC or2-amino purine deoxyriboside is another analog that could be used inprobe synthesis. The probes containing such nucleotide derivatives maybe hydrolyzed to release much more strongly fluorescent mononucleotidesby flap-specific nuclease activity.

C. Cleaving a Cleavage Structure and Generating a Signal

A cleavage structure according to the invention can be cleaved by themethods described in the section above, entitled “FEN Nucleases”.

D. Detection of Released Labeled Fragments

Detection or verification of the labeled fragments may be accomplishedby a variety of methods well known in the art and may be dependent onthe characteristics of the labeled moiety or moieties comprising alabeled cleavage structure.

In one embodiment of the invention, the reaction products, including thereleased labeled fragments, are subjected to size analysis. Methods fordetermining the size of a labeled fragment are known in the art andinclude, for example, gel electrophoresis, sedimentation in gradients,gel exclusion chromatography, mass spectroscopy, and homochromatography.

During or after amplification, separation of the released labeledfragments from, for example, a PCR mixture can be accomplished by, forexample, contacting the PCR with a solid phase extractant (SPE). Forexample, materials having an ability to bind nucleic acids on the basisof size, charge, or interaction with the nucleic acid bases can be addedto the PCR mixture, under conditions where labeled, uncleaved nucleicacids are bound and short, labeled fragments are not. Such SPE materialsinclude ion exchange resins or beads, such as the commercially availablebinding particles Nensorb (DuPont Chemical Co.), Nucleogen (The NestGroup), PEI, BakerBond™ PEI, Amicon PAE 1,000, Selectacel™ PEI, BoronateSPE with a 3′-ribose probe, SPE containing sequences complementary tothe 3′-end of the probe, and hydroxylapatite. In a specific embodiment,if a dual labeled oligonucleotide comprising a 3′ biotin label separatedfrom a 5′ label by a nuclease susceptible cleavage site is employed asthe signal means, the reaction mixture, for example a PCR amplifiedmixture can be contacted with materials containing a specific bindingpartner such as avidin or streptavidin, or an antibody or monoclonalantibody to biotin. Such materials can include beads and particlescoated with specific binding partners and can also include magneticparticles.

Following the step in which a reaction mixture, for example a PCRmixture has been contacted with an SPE, the SPE material can be removedby filtration, sedimentation, or magnetic attraction, leaving thelabeled fragments free of uncleaved labeled oligonucleotides andavailable for detection.

IV. Methods of Use

The invention provides for a method of generating a signal indicative ofthe presence of a target nucleic acid sequence in a sample comprisingthe steps of forming a labeled cleavage structure by incubating a targetnucleic acid sequence with a nucleic acid polymerase, and cleaving thecleavage structure with a FEN nuclease. The method of the invention canbe used in a PCR based assay as described below.

A labeled cleavage structure comprising an upstream oligonucleotideprimer (for example A, FIG. 3), a 5′ end labeled downstreamoligonucleotide probe (for example C in FIG. 3) and a target nucleicacid sequence (for example B in FIG. 3) is formed as described above inthe section entitled “Cleavage Structure”. Briefly, a cleavage structureis formed and cleaved in the presence of a target nucleic acid sequence,an upstream primer (for example A, FIG. 3), a labeled downstream probe(for example C, FIG. 3) amplification primers specific for the targetnucleic acid sequence, a nucleic acid polymerase deficient in 5′ to 3′exonuclease activity, a FEN nuclease and an appropriate buffer (forexample 10×Pfu buffer, Stratagene, Catalog #200536) in a PCR reactionwith the following thermocycling parameters: 95° C. for 2 minutes and 40cycles of 95° C. for 15 sec (denaturation step), 60° C. for 60 sec(annealing step) and 72° C. for 15 sec (extension step). During thisreaction an upstream oligonucleotide (for example A, FIG. 3) is extendedsuch that oligonucleotide A partially displaces the 5′ labeled end of adownstream oligonucleotide that is annealed to a target nucleic acidsequence (for example oligonucleotide C, FIG. 3) and the resultinglabeled structure is cleaved with a FEN nuclease according to theinvention.

The methods of the invention can also be used in non-PCR basedapplications to detect a target nucleic acid sequence, where such targetthat may be immobilized on a solid support. Methods of immobilizing anucleic acid sequence on a solid support are known in the art and aredescribed in Ausubel F M et al. Current Protocols in Molecular Biology,John Wiley and Sons, Inc. and in protocols provided by themanufacturers, e.g. for membranes: Pall Corporation, Schleicher &Schuell, for magnetic beads: Dynal, for culture plates: Costar,Nalgenunc, and for other supports useful according to the invention,CPG, Inc. A solid support useful according to the invention includes butis not limited to silica based matrices, membrane based matrices andbeads comprising surfaces including, but not limited to styrene, latexor silica based materials and other polymers. Magnetic beads are alsouseful according to the invention. Solid supports can be obtained fromthe above manufacturers and other known manufacturers.

The invention also provides for a non-PCR based assay for detecting atarget nucleic acid sequence in solution. The method of the inventioncan be used to detect naturally occurring target nucleic acid sequencesin solution including but not limited to RNA and DNA that is isolatedand purified from cells, tissues, single cell organisms, bacteria orviruses. The method of the invention can also be used to detectsynthetic targets in solution, including but not limited to RNA or DNAoligonucleotides, and peptide nucleic acids (PNAs). Non-PCR assaysinclude but are not limited to detection assays involving isothermallinear or exponential amplification, where the amount of nucleic acidsynthesized by the 3′-5′ synthetic activity increases linearly orexponentially, and a FEN nuclease is used to cleave the displaced strandduring synthesis. One such example utilizes rolling circleamplification.

Detection of a nucleic acid target sequence that is either immobilizedor in solution can be performed by incubating an immobilized nucleicacid target sequence or a target nucleic acid sequence in solution withan upstream oligonucleotide primer that is complementary to the targetnucleic acid sequence (for example A, FIG. 3) and a downstreamoligonucleotide probe that is complementary to the target nucleic acidsequence (for example C, FIG. 3), a FEN nuclease and a nucleic acidpolymerase lacking 5′ to 3′ exonuclease activity. The downstream probeis either end labeled at the 5′ or 3′ end, or is labeled internally.Detection of a released labeled fragment involves isotopic, enzymatic,or colorimetric methods appropriate for the specific label that has beenincorporated into the probe. Labels useful according to the inventionand methods for the detection of labels useful according to theinvention are described in the section entitled “Cleavage Structure”.Alternatively, the downstream probe comprises a pair of interactivesignal generating labeled moieties (for example a dye and a quencher)that are positioned such that when the probe is intact, the generationof a detectable signal is quenched, and wherein the pair of interactivesignal generating moieties are separated by a FEN nuclease cleavagesite. Upon cleavage by a FEN nuclease, the two signal generatingmoieties are separated from each other and a detectable signal isproduced. Nucleic acid polymerases that are useful for detecting animmobilized nucleic acid target sequence or a nucleic acid targetsequence in solution according to the method of the invention includemesophilic, thermophilic or hyper-thermophilic DNA polymerases lacking5′ to 3′ exonucleolytic activity (described in the section entitled,“Nucleic Acid Polymerases)”.

According to this non-PCR based method, the amount of a target nucleicacid sequence that can be detected is preferably about 1 pg to 1

g, more preferably about 1 pg to 10 ng and most preferably about 1 pg to10 pg. Alternatively, this non-PCR based method can measure or detectpreferably about 1 molecule to 10²⁰ molecules, more preferably about 100molecules to 10¹⁷ molecules and most preferably about 1000 molecules to10¹⁴ molecules.

The invention also provides for a method of detecting a target nucleicacid sequence in a sample wherein a cleavage structure is formed asdescribed in the section entitled, “Cleavage Structure”, and the targetnucleic acid sequence is amplified by a non-PCR based method includingbut not limited to an isothermal method, for example rolling circle,Self-sustained Sequence Replication Amplification (3SR), Transcriptionbased amplification system (TAS), and Strand Displacement Amplification(SDA) and a non-isothermal method, for example Ligation chain reaction(LCR). A FEN nuclease useful for non-PCR amplification methods will beactive at a temperature range that is appropriate for the particularamplification method that is employed.

In the amplification protocols described below, samples which need to beprepared in order to quantify the target include: samples, no-templatecontrols, and reactions for preparation of a standard curve (containingdilutions over the range of six orders of magnitude of a solution with adefined quantity of target).

Strand Displacement Amplification (SDA) is based on the ability of arestriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its recognition site. The appropriate DNApolymerase will initiate replication at this nick and displace thedownstream non-template strand (Walker, 1992, Proc. Natl. Acad. Sci.USA, 89: 392, and PCR Methods and Applications 3: 1-6, 1993). Thepolymerases (Bca and Bst) which are used according to the method of SDAcan also be used in FEN directed cleavage according to the invention.According to the method of the invention, a molecular beacon is replacedby a FEN nuclease active at 420 C and a cleavable probe comprising acleavage structure according to the invention.

A molecular beacon (Mb) is a fluorogenic probe which forms a stem-loopstructure is solution. Typically: 5′-fluorescent dye (e.g. FAM),attached to the 5′-stem region (5-7 nt), the loop region (complementaryto the target, 20 to 30 nt), the 3′-stem region (complementary to the5′-stem region), and the quencher (e.g. DABCYL). If no target ispresent, the MB forms its stem, which brings dye and quencher into closeproximity, and therefore no fluorescence is emitted. When an MB binds toits target, the stem is opened, dye is spatially separated from thequencher, and therefore the probe emits fluorescence (Tyagi S and KramerF R, Nature Biotechnology 14: 303-308 (1996) and U.S. Pat. No.5,925,517).

Strand Displacement Amplification (SDA) is essentially performed asdescribed by Spargo et al., Molecular and Cellular Probes 10: 247-256(1996). The enzymes used include restriction endonuclease BsoBI (NewEngland Biolabs), DNA polymerase 5′-exo-Bca (PanVera Corporation). Thetarget is an insertion-like element (IS6110) found in the Mycobacteriumtuberculosis (Mtb) genome. The primers used are B1: cgatcgagcaagcca (SEQID NO: 4), B2: cgagccgctcgctg (SEQ ID NO: 5), S1:accgcatcgaatgcatgtctcgggtaaggcgtactcgacc (SEQ ID NO: 6) and S2:cgattccgctccagacttctcgggtgtactgagatcccct (SEQ ID NO: 7). TheMycobacterium tuberculosis genomic DNA is serially diluted in humanplacental DNA. SDA is performed in 50 ul samples containing 0 to 1000Mtb genome equivalents, 500 ng human placental DNA, 160 units BsoB1, 8units of 5′-exo-Bca, 1.4 mM each dCTPalphaS, TTP, dGTP, dATP, 35 mMK₂PO₄, pH 7.6 0.1 mg/ml acetylated bovine serum albumin (BSA), 3 mMTris-HCl, 10 mM MgCl₂, 11 mM NaCl, 0.3 mM DTT, 4 mM KCl, 4% glycerol,0.008 mM EDTA, 500 nM primers S1 and S2 and 50 nM primers B1 and B2(KCl, glycerol and EDTA are contributed by the BsoB1 storage solution).The samples (35 μl) were heated in a boiling water bath for 3 minutesbefore the addition of BsoB1 and 5′-exo Bca (10.7 units/μl BsoB1 and0.53 units/μl 5′-exo Bca in 15 μl of New England Biolabs Buffer 2 (20 mMTris-HCl pH 7.9, 10 mM MgCl₂, 50 mM NaCl, 1 mM DTT). Incubation is at60° C. for 15 minutes, followed by 5 minutes in a boiling water bath.

Five μl of each sample in duplicate are removed for detection. Eachreaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂, 200 uM of eachdNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer: aaggcgtactcgacctgaaa (SEQ ID NO: 8) and fluorogenic probe (forexample FAM-DABCYL): accatacggataggggatctc (SEQ ID NO: 9). The reactionsare subjected to one cycle in a thermal cycler: 2 minutes at 95° C., 1minute at 55° C., 1 minute at 72° C. The fluorescence is then determinedin a fluorescence plate reader, such as Stratagene's FluorTracker or PEBiosystems' 7700 Sequence Detection System in Plate-Read Mode.

According to the method of nucleic acid sequence-based amplification(NASBA), molecular beacons are used for quantification of the NASBA RNAamplicon in real-time analysis (Leone, et al., 1998, Nucleic Acids Res.26: 2150). According to the method of the invention, NASBA can becarried out wherein the molecular beacon probe is replaced by aFENnuclease cleavable probe comprising a cleavage structure according tothe invention and a FEN nuclease active at 41° C.

NASBA amplification is performed essentially as described by Leone G, etal., Nucleic Acids Res. 26: 2150-2155 (1998). Genomic RNA from thepotato leafroll virus (PLRV) is amplified using the PD415 or PD416(antisense) and the PD417 (sense) primers, which are described in detailin Leone G et al., J. Virol. Methods 66: 19-27 (1997). Each NASBAreaction contains a premix of 6

l of sterile water, 4 μl of 5×NASBA buffer (5×NASBA buffer is 200 mMTris-HCl, pH 8.5, 60 mM MgCl₂, 350 mM KCl, 2.5 mM DTT, 5 mM each ofdNTP, 10 mM each of ATP, UTP and CTP, 7.5 mM GTP and 2.5 mM ITP), 4 μlof 5× primer mix (75% DMSO and 1 μM each of antisense and senseprimers). The premix is divided into 14 μl aliquots, to which 1 μl ofPLRV target is added. After incubation for 5 minutes at 65° C. andcooling to 41° C. for 5 minutes, 5 μl of enzyme mix is added (perreaction 375 mM sorbitol, 2.1 μg BSA, 0.08 units of RNase H (Pharmacia),32 units of T7 RNA polymerase (Pharmacia) and 6.4 units of AMV-RT(Seigakaku)). Amplification is for 90 minutes at 41° C.

Five μl of each sample in duplicate are removed for detection. Eachreaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂, 200 uM of eachdNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer PD415 or PD416 and the fluorogenic probe (for exampleFAM-DABCYL): gcaaagtatcatccctccag (SEQ ID NO: 10). The reactions aresubjected to one cycle in a thermal cycler: 2 minutes at 95° C., 1minute at 55° C., 1 minute at 72° C. The fluorescence in then determinedin a fluorescence plate reader, such as Stratagene's FluorTracker or PEBiosystems' 7700 Sequence Detection System in Plate-Read Mode.

Generally, according to these methods wherein amplification occurs by anon-PCR based method, amplification may be carried out in the presenceof a FEN nuclease, and amplification and cleavage by the FEN nucleaseoccur simultaneously. Detection of released labeled fragments isperformed as described in the section entitled “Cleavage Structure” andmay occur concurrently with (real time) or after (end-point) theamplification and cleavage process have been completed.

Endpoint assays can be used to quantify amplified target produced bynon-PCR based methods wherein the amplification step is carried out inthe presence of a FEN nuclease (described above).

Endpoint assays include, but are not limited to the following.

A. Ligation chain reaction (LCR), as described in Landegren, et al.,1988, Science, 241: 1077 and Barany, PCR Methods and Applications 1:5-16 (1991). An LCR product useful according to the invention will belong enough such that the upstream primer and the labeled downstreamprobe are separated by a gap larger than 8 nucleotides to allow forefficient cleavage by a FEN nuclease.

B. Self-sustained sequence replication amplification (3SR) Fahy, et al.PCR Methods and Applications 1: 25-33 (1991). Self-Sustained SequenceReplication Amplification (3SR) is a technique which is similar toNASBA. Ehricht R, et al., Nucleic Acids Res. 25: 4697-4699 (1997) haveevolved the 3SR procedure to a cooperatively coupled in vitroamplification system (CATCH). Thus, in CATCH, a molecular beacon probeis used for real-time analysis of an RNA amplicon. The synthetic targetamplified has the sequence: cctctgcagactactattacataatacgactcactatagggatctgcacgtattagcctatagtgagtcgtattaataggaaacaccaaagatgatatttcgtcacagcaagaattcagg(SEQ ID NO: 11). The 3SR reactions contain 40 mM Tris-HCl pH 8.0, 5 mMKCl, 30 mM MgCl₂, 1 mM of each dNTP, 1 nM of the double stranded target,2 μM P1: cctctgcagactactattac (SEQ ID NO: 12) andP2:cctgaattcttgctgtgacg (SEQ ID NO: 13), 5 mM DTT, 2 mM spermidine, 6units/ul His tagged HIV-1 reverse transcriptase, 3 units/ul T7-RNApolymerase and 0.16 units/ul Escherichia coli RNase H. The 100 ulreactions are incubated for 30 minutes at 42° C.

Five μl of each sample in duplicate are removed for detection. Eachreaction contains 1× Cloned Pfu buffer, 3.0 mM MgCl₂, 200 uM of eachdNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 300 nM each upstreamprimer P1 and fluorogenic probe (for example FAM-DABCYL):taggaaacaccaaagatgatattt (SEQ ID NO: 14). The reactions are subjected toone cycle in a thermal cycler: 2 minutes at 95° C., 1 minute at 55° C.,1 minute at 72° C. The fluorescence in then determined in a fluorescenceplate reader, such as Stratagene's FluorTracker or PE Biosystems' 7700Sequence Detection System in Plate-Read Mode.

C. Rolling circle amplification is described in U.S. Pat. No. 5,854,033and the related Ramification-Extension Amplification Method (RAM) (U.S.Pat. No. 5,942,391). Rolling circle amplification adapted to theinvention is described in Example 3 below.

Real-time assays can also be used to quantify amplified target producedby non-PCR based methods wherein the amplification step is carried outin the presence of a FEN nuclease (described above). The method ofrolling circle amplification (U.S. Pat. No. 5,854,033) is adapted toinclude secondary primers for amplification and detection, inconjunction with a FEN nuclease and a cleavable probe comprising acleavage structure according to the invention and is carried out attemperatures between 50-60° C.

The cleavage pattern of a FEN nuclease can be altered by the presence ofa single mismatched base located anywhere between 1 and 15 nucleotidesfrom the 5′ end of the primer wherein the DNA primer is otherwise fullyannealed. Typically, on a fully annealed substrate, a FEN nuclease willexonucleolytically cleave the 5′ most nucleotide. However, a singlenucleotide mismatch up to 15 nucleotides in from the 5′ end promotesendonucleolytic cleavages. This constitutes a 5′ proofreading process inwhich the mismatch promotes the nuclease action that leads to itsremoval. Thus, the mechanism of FEN nuclease cleavage is shifted frompredominantly exonucleolytic cleavage to predominantly endonucleolyticcleavage simply by the presence of a single mismatched base pair.Presumably this occurs because a mismatch allows a short flap to becreated (Rumbaugh et al., 1999, J. Biol. Chem., 274:14602).

The method of the invention can be used to generate a signal indicativeof the presence of a sequence variation in a target nucleic acidsequence, wherein a labeled cleavage structure comprising a fullyannealed DNA primer is formed by incubating a target nucleic acidsequence with a nucleic acid polymerase (as described in the sectionentitled, “Cleavage Structure”) and cleaving the labeled cleavagestructure with a FEN nuclease wherein the release of labeled fragmentscomprising endonucleolytic cleavage products is indicative of thepresence of a sequence variation. Released labeled fragments aredetected as described in the section entitled, “Cleavage Structure”.

V. Samples

The invention provides for a method of detecting or measuring a targetnucleic acid sequence in a sample, as defined herein. As used herein,“sample” refers to any substance containing or presumed to contain anucleic acid of interest (a target nucleic acid sequence) or which isitself a target nucleic acid sequence, containing or presumed to containa target nucleic acid sequence of interest. The term “sample” thusincludes a sample of target nucleic acid sequence (genomic DNA, cDNA orRNA), cell, organism, tissue, fluid or substance including but notlimited to, for example, plasma, serum, spinal fluid, lymph fluid,synovial fluid, urine, tears, stool, external secretions of the skin,respiratory, intestinal and genitourinary tracts, saliva, blood cells,tumors, organs, tissue, samples of in vitro cell culture constituents,natural isolates (such as drinking water, seawater, solid materials,)microbial specimens, and objects or specimens that have been “marked”with nucleic acid tracer molecules.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1

A target nucleic acid sequence can be detected and/or measured by thefollowing method. A labeled cleavage structure is formed prior to theaddition of a FEN nuclease by heating at 950 C for 5 minutes and thencooling to approximately 50-60° C. (a) a sample containing a targetnucleic acid sequence (B in FIG. 3) with (b) an upstream oligonucleotidethat specifically hybridizes to the target nucleic acid sequence, (A, inFIG. 3), and (c) a downstream, 5′ end labeled oligonucleotide (C in FIG.3) that specifically hybridizes to a region of the target nucleic acidsequence that is downstream of the hybridizing region of oligonucleotideA. A polymerase that lacks a 5′ to 3′ exonuclease activity but thatpossesses a 3′ to 5′ DNA synthetic activity, such as the enzyme a) Yaqexo-, (prepared by mutagenesis using the Stratagene QuikChangeSite-Directed Mutagenesis kit, catalog number #200518, to modify Taqpolymerase (Tabor and Richardson, 1985, Proc. Natl. Acad. Sci. USA,82:1074)), a mutant form of Taq polymerase that lacks 5′ to 3′exonuclease activity, b) Pfu, or c) a mutant form of Pfu polymerase thatlacks 3′ to 5′ exonuclease activity (exo-Pfu) is added and incubatedunder conditions that permit the polymerase to extend oligonucleotide Asuch that it partially displaces the 5′ end of oligonucleotide C (forexample 720 C in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour. Thedisplaced region of oligonucleotide C forms a 5′ flap that is cleavedupon the addition of a FEN nuclease.

A mutant form of Taq polymerase that lacks a 5′ to 3′ exonucleaseactivity but that possesses a 3′ to 5′ DNA synthetic activity comprisesthe following mutation: D144S/F667Y Taq wherein D144S eliminates 5′ to3′ exonuclease activity and F667Y improves ddNTP incorporation.

Exo-mutants of PolI polymerase can be prepared according to the methodof Xu et al., 1997, J. Mol. Biol., 268: 284.

A labeled cleavage structure according to the invention is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 2). Cleavage is carried out byadding 2 μl of PfuFEN-1 to a 7 μl reaction mixture containing thefollowing:

  3 μl cleavage structure (10 ng-10 μg)  0.7 μl 10x FEN nuclease buffer(10X FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O  1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution (included in the Stratagene Cyclist DNA sequencing kit, catalog#200326, and described in example 3), samples are heated at 99° C. forfive minutes. Samples are loaded on an eleven inch long, hand-poured,20% acrylamide/bis acrylamide, 7M urea gel. The gel is run at 20 wattsuntil the bromophenol blue has migrated approximately ⅔ the totaldistance. The gel is removed from the glass plates and soaked for 10minutes in fix solution (15% methanol, 5% acetic acid) and then for 10minutes in water. The gel is placed on Whatmann 3 mm paper, covered withplastic wrap and dried for 2 hours in a heated vacuum gel dryer (˜80°C.). The gel is exposed overnight to X-ray film to detect the presenceof a signal that is indicative of the presence of a target nucleic acidsequence.

Example 2 Cloning Pfu FEN-1

A thermostable FEN nuclease enzyme useful according to the invention canbe prepared according to the following method.

The thermostable FEN nuclease gene can be isolated from genomic DNAderived from P. furiosus (ATCC#43587) according to methods of PCRcloning well known in the art. The cloned PfuFEN-1 can be overexpressedin bacterial cells according to methods well known in the art anddescribed below.

The following pCAL-n-EK cloning oligonucleotides were synthesized andpurified:

(SEQ ID NO: 15) a. 5′GACGACGACAAGATGGGTGTCCCAATTGGTGAGATTATACCAAGAAAA G3′ and (SEQ ID NO: 16) b.5′GGAACAAGACCCGTTTATCTCTTGAACCAACTTTCAAGGGTTGATTGT TTTCCACT 3′.

The Affinity® Protein Expression and Purification System was obtainedfrom Stratagene and used according to the manufacturer's protocols.

Amplification

The insert DNA was prepared by PCR amplification with gene-specificprimers (oligonucleotides a and b, described above) that include 12 and13-nucleotide sequences at the 5′ ends that are complementary to thepCAL-n-EK vector single-stranded tails, thus allowing for directionalcloning. The FEN-1 sequence was amplified from genomic DNA derived fromP. furiosus by preparing amplification reactions (five independent 100μl reactions) containing:

 50 μl 10x cPfu Buffer (Stratagene)  7.5 μl Pfu Genomic DNA (approx. 100ng/μl)  7.5 μl PfuTurbo(2.5 u/μl), (Stratagene, Catalog # 600250)  15 μlmixed primer pair (100 ng/μl each) (oligonucleotides a and b, describedabove)  4 μl 100 mM dNTP 416 μl H₂O 500 μl totaland carrying out the amplification under the following conditions usinga Stratagene Robocycler 96 hot top thermal cycler:

Window 1 95° C. 1 minute  1 cycle Window 2 95° C. 1 minute 50° C. 1minute 30 cycles 72° C. 3 minutes

The PCR products from each of the five reactions were combined into onetube, purified using StrataPrep PCR and eluted in 50

l 1 mM Tris-HCl pH 8.6. The FEN-1 PCR product was analyzed on a gel andwas determined to be approximately 1000 bp.

The PCR product comprising the fen-1 gene was cloned into the pCALnEKLIC vector (Stratagene) by creating ligation independent cloning termini(LIC), annealing the PCR product comprising the fen-1 gene to thepCALnEK LIC vector (Stratagene), and transforming cells with theannealing mixture according to the following method. Briefly, followingPCR amplification, the PCR product is purified and treated with Pfu DNApolymerase in the presence of dATP (according to the manual includedwith the Affinity® Protein Expression and Purification System,Stratagene, catalog #200326). In the absence of dTTP, dGTP and dCTP, the3′ to 5′-exonuclease activity of Pfu DNA polymerase removes at least 12and 13 nucleotides at the respective 3′ ends of the PCR product. Thisactivity continues until the first adenine is encountered, producing aDNA fragment with 5′-extended single-stranded tails that arecomplementary to the single-stranded tails of the pCAL-n-EK vector.

Creating LIC Termini

LIC termini were created by preparing the following mixture:

 45 μl purified PCR product (~0.5 μg/μl) 2.5 μl 10 mM dATP   5 μl 10xcPfu buffer   1 μl cPfu (2.5 u/μl) 0.5 μl H₂O

cPfu and cPfu buffer can be obtained from Stratagene (cPfu, StratageneCatalog #600153 and cPfu buffer, Stratagene Catalog #200532).

Samples were incubated at 72° C. for 20 minutes and products were cooledto room temperature. To each sample was added 40 ng prepared pCALnEK LICvector (the prepared vector is available commercially from Stratagene inthe Affinity LIC Cloning and Protein Purification Kit (214405)). Thevector and insert DNA are combined, allowed to anneal at roomtemperature and transformed into highly competent bacterial host cells(Wyborski et al., 1997, Strategies, 10:1).

Preparing Cells for Production of FEN

Two liters of LB-AMP was inoculated with 20 ml of an overnight cultureof a FEN-1 clone (clone 3). Growth was allowed to proceed forapproximately 11 hours at which point cells had reached an OD₆₀₀=0.974.Cells were induced overnight (about 12 hours) with 1 mM IPTG. Cells werecollected by centrifugation and the resulting cell paste was stored at−20° C.

Purification of Tagged FEN-1

Cells were resuspended in 20 ml of Calcium binding buffer

CaCl₂ Binding Buffer

50 mM Tris-HCl (pH 8.0)

150 mM NaCl

1.0 mM MgOAc

2 mM CaCl₂

The samples were sonicated with a Branson Sonicator using a microtip.The output setting was 5 and the duty cycle was 90%. Samples weresonicated three times and allowed to rest on ice during the intervals.The sonicate was centrifuged at 26,890×g. Cleared supernatants weremixed with 1 ml of washed (in CaCl₂ binding buffer) calmodulin agarose(CAM agarose) in a 50 ml conical tube and incubated on a slowly rotatingwheel in a cold room (40 C) for 5 hours. The CAM agarose was collectedby light centrifugation (5000 rpm in a table top centrifuge).

Following removal of the supernatant, the CAM agarose was washed with 50ml CaCl₂ binding buffer and transferred to a disposable drip column. Theoriginal container and pipet were rinsed thoroughly to remove residualagarose. The column was rinsed with approximately 200 ml of CaCl₂binding buffer.

Elution was carried out with 10 ml of 50 mM NaCl elution buffer (50 mMNaCl, 50 mM Tris-HCl pH 8.0, 2 mM EGTA). 0.5 ml fractions werecollected. A second elution step was carried out with 1M NaCl elutionbuffer wherein 0.5 ml fractions were collected.

Evaluation of Purified Tagged FEN-1

Fractions containing CBP-tagged Pfu FEN-1 eluted in 1M NaCl were boiledin SDS and analyzed by SDS-PAGE on a 4-20% gel stained with Sypro Orange(FIG. 4).

The protein concentration of uncleaved FEN-1 was determined to beapproximately 150 ng/microliter (below).

Enterokinase Protease (EK) Cleavage of the Purified FEN-1

Fractions 3-9 were dialyzed in 50 mM NaCl, 50 mM Tris-HCl pH 8.0 and 2mM CaCl₂ overnight at 4° C.

An opaque, very fine precipitate appeared in the dialyzed FEN-1. Whenthe sample was diluted 1/20 the precipitate was removed. When the samplewas diluted 1/3 insoluble material was still detectable. The 1/3 dilutedmaterial was heated at 37° C. for 2 minutes and mixed with Tween 20 to afinal concentration of 0.1%. Upon the addition of the Tween 20, therewas an almost immediate formation of “strings” and much coarser solidsin the solution which could not be reversed even after the solution wasadjusted to 1M NaCl.

EK cleavage was carried out using as a substrate the sample that wasdiluted 1/20 as well as with a dilute sample prepared by rinsing thedialysis bag with 1×EK buffer. EK cleavage was carried out by theaddition of 1 μl EK (1 u/μl) overnight at room temperature (about 16hours).

100 μl of STI agarose combined with 100 μl of CAM agarose were rinsedtwice with 10 ml of 1×STI buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 2mM CaCl₂, 0.1% Tween 20). NaCl was added to the two EK samples to bringthe final concentration to 200 mM NaCl. The two samples were combinedand added to the rinsed agarose. The samples were rotated slowly on awheel at 4° C. for three hours and separated by light centrifugation ina table top centrifuge (as described). The supernatant was removed andthe resin was rinsed twice with 500 μl 1×STI. The two rinses werecombined and saved separately from the original supernatant. Sampleswere analyzed by SDS-PAGE on a 4-20% gel.

The concentration of digested product was approximately 23 ng/μl asdetermined by comparison to a Pfu standard at a concentration ofapproximately 50 ng/ml.

Example 3 FEN Nuclease Activity

The endonuclease activity of a FEN nuclease and the cleavage structurerequirements of a FEN nuclease prepared as described in Example 2 can bedetermined according to the methods described either in the sectionentitled “FEN nucleases” or below.

Briefly, three templates (FIG. 2) are used to evaluate the activity of aFEN nuclease according to the invention. Template 1 is a 5′ ³³P labeledoligonucleotide (Heltest4) with the following sequence:

(SEQ ID NO: 1) 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′.

The underlined section of Heltest4 represents the region complementaryto M13mp18+. The cleavage product is an 18 nucleotide fragment with thesequence AAAATAAATAAAAAAAAT (SEQ ID NO: 2). Heltest4 binds to M13 toproduce a complementary double stranded domain as well as anon-complementary 5′ overhang. This duplex forms template 2 (FIG. 2).Template 3 (FIG. 2) has an additional primer (FENAS) bound to M13 whichis directly adjacent to Heltest 4. The sequence of FENAS is:

5′ CCATTCGCCATTCAGGCTGCGCA 3′ (SEQ ID NO: 3). In the presence oftemplate 3, a FEN nuclease binds the free 5′ terminus of Heltest4,migrates to the junction and cleaves Heltest4 to produce an 18nucleotide fragment. The resulting cleavage products are separated on a6% acrylamide, 7M urea sequencing gel.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl 

Pfu buffer can be obtained from Stratagene (Catalog #200536).

The template mixture is heated at 95° C. for five minutes, cooled toroom temperature for 45 minutes and stored at 4° C. overnight.

The enzyme samples are as follows:

A. H₂O (control) B. 2 μl undiluted uncleaved FEN-1 (~445 ng/μl) C. 2 μl1/10 dilution of uncleaved FEN-1 (~44.5 ng/μl) D. 2 μl enterokinaseprotease (EK) cleaved FEN-1 (~23 ng/μl)

The four reaction mixtures are mixed with the three templates asfollows:

  3 μl template 1, template 2 or template 3  0.7 μl 10x cloned Pfubuffer  0.6 μl 100 mM MgCl₂ 2.00 μl FEN-1 or H₂O  0.7 μl H₂O 7.00 μltotal volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide 7M urea CastAway gel (Stratagene).

Alternatively, FEN nuclease activity can be analyzed in the followingbuffer wherein a one hour incubation time is utilized.

10×FEN Nuclease Buffer

500 mM Tris-HCl pH 8.0

100 mM MgCl₂

The reaction mixture is as follows:

  3 μl template 1, template 2 or template 3  0.7 μl 10X FEN nucleasebuffer 2.00 μl FEN-1 or H₂O (A-D, above)  1.3 μl H₂O 7.00 μl totalvolume

Samples are incubated for one hour at 50° C. in the Robocyler 96 hot topthermal cycler. Following the addition of 2

l of Sequencing Stop (95% formamide, 20 mM EDTA, 0.05% bromophenol blue,0.05% xylene cyanol, available from Stratagene) dye solution, samplesare heated at 99° C. for five minutes. Samples are loaded on an eleveninch long, hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. Thegel is run at 20 watts until the bromophenol blue has migratedapproximately ⅔ the total distance. The gel is removed from the glassplates and soaked for 10 minutes in fix solution (15% methanol, 5%acetic acid) and then for 10 minutes in water. The gel is placed onWhatmann 3 mm paper, covered with plastic wrap and dried for 2 hours ina heated vacuum gel dryer (˜800 C). The gel is exposed overnight toX-ray film.

An autoradiograph of a FEN-1 nuclease assay wherein templates 1, 2 and 3(prepared as described above) are cleaved by the addition of:

A. H₂O B. 2 μl of CBP-tagged Pfu FEN-1 C. 2 μl of CBP-tagged Pfu FEN-1diluted (1:10) D. 2 μl of EK cleaved Pfu FEN-1is presented in FIG. 5.

The lanes are as follows. Lanes 1A, 1B, 1C and 1D represent template 1cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10 dilution ofCBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively. Lanes 2A,2B, 2C and 2D represent template 2 cleaved with H₂O, undilutedCBP-tagged Pfu FEN-1, a 1:10 dilution of CBP-tagged Pfu FEN-1 and EKcleaved Pfu FEN-1, respectively. Lanes 3A, 3B, 3C and 3D representtemplate 3 cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10dilution of CBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively.

Tagged Pfu FEN-1 contains the N-terminal CBP affinity purification tag.Any differences in activity between tagged and untagged versions ofFEN-1 are due to differences in protein concentration (concentrations ofenzyme samples are provided above) since the amounts of tagged versusuntagged FEN-1 are not equivalent. Both tagged and untagged Pfu FEN-1demonstrate cleavage activity.

FIG. 5 demonstrates the background level of cleavage in the absence ofFEN-1 (lanes 1A, 2A and 3A). Further, this figure demonstrates thattagged Pfu FEN-1 cleaves more of template 2 as compared to template 1.In particular, the greatest amount of template 2 is cleaved in thepresence of undiluted, tagged Pfu FEN-1 (lane 2B). Analysis of template3 demonstrates that the greatest amount of template 3 is cleaved byundiluted, tagged Pfu FEN-1 and the least amount of template 3 iscleaved by diluted tagged FEN-1. Labeled probe migrates as a 40-43nucleotide band. FEN-1 preferentially cleaves template 3 (whichcomprises an upstream primer) as compared to template 2. The cleavageproduct bands are the major bands migrating at 16-20 nucleotides.Heterogeneity in the labeled cleavage products is the result ofheterogeneity in the labeled substrate, which was not gel-purified priorto use.

Example 4 PCR Amplification and Detection of β-Actin in the Presence ofa FEN-1 Nuclease and a Tag Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

A PCR assay is used to detect a target nucleic acid sequence. Accordingto the method of this assay, a PCR reaction is carried out in thepresence of a Taq polymerase deficient in 5′ to 3′ exonuclease activity(for example Yaq exo-), and a thermostable FEN-1 nuclease (e.g. PfuFEN-1, prepared as described in Example 2). Detection of the release offluorescently labeled fragments indicates the presence of the targetnucleic acid sequence.

Duplicate PCR reactions containing 1× Sentinel Molecular beacon corebuffer, 3.5 mM MgCl₂, 200 μM of each dNTP, a Taq polymerase deficient in5′ to 3′ exonuclease activity (˜1.45 U), Pfu FEN-1 (˜23 ng), PEBiosystems β-Actin primers (300 nM each) (CATALOG #600500) and β-actinspecific fluorogenic probe (200 nM; 5′ FAM−3′TAMRA+PE Biosystems catalog#P/N 401846) were prepared. 10 ng of human genomic DNA (Promega) wasused as the target nucleic acid sequence in each reaction. This reactionwas performed in a 50 μl volume. Negative control reactions containingeither Pfu FEN-1 alone, a Taq polymerase deficient in 5′ to 3′exonuclease activity alone or reaction mixtures containing allcomponents except a human genomic DNA template were prepared. Positivecontrol reactions comprising 2.5 Units of Taq 2000 were also prepared.Reactions were assayed in a spectrofluorometric thermocycler (ABI 7700).Thermocycling parameters were 95° C. for 2 min and 40 cycles of 95° C.for 15 sec, 60° C. for 60 sec and 72° C. for 15 sec. Samples wereinterrogated during the annealing step.

As demonstrated in FIG. 6, no signal was generated in the presence ofeither Pfu FEN-1 alone or a Taq polymerase deficient in 5′ to 3′exonuclease activity alone. In the presence of both a Taq polymerasedeficient in 5′ to 3′ exonuclease activity and Pfu FEN-1 a signal wasgenerated with a threshold cycle (Ct) of 26 and a final fluorescenceintensity (FI) of 12,000 units. In the presence of Taq 2000 (a nucleicacid polymerase which has 5′ to 3′ exonuclease activity) (Taqman) asignal was generated with a Ct of 23 and a FI of 17,000 units.

These results demonstrate that β-actin DNA sequences can be detected bya PCR assay wherein a signal is generated in the presence of a Taqpolymerase deficient in 5′ to 3′ exonuclease activity and a thermostableFEN-1 nuclease. Further, the 5′ to 3′ exonuclease activity that isabsent in the Taq polymerase deficient in 5′ to 3′ exonuclease activitycan be restored, in trans by the addition of Pfu FEN-1.

Example 5 PCR Amplification and Detection of β-Actin in the Presence ofa FEN-1 Nuclease and a Pfu Polymerase Deficient in 5′ to 3′ ExonucleaseActivity

A PCR assay is used to detect a target nucleic acid sequence. Accordingto the method of this assay, a PCR reaction is carried out in thepresence of a Pfu polymerase (naturally lacking 5′ to 3′ exonucleaseactivity) or, in addition, Pfu polymerase deficient in 3′ to 5′exonuclease activity as well (for example exo-Pfu), and a thermostableFEN-1 nuclease (Pfu FEN-1). Detection of the release of fluorescentlylabeled fragments indicates the presence of the target nucleic acidsequence.

Duplicate PCR reactions containing 1× Cloned Pfu buffer (available fromStratagene, Catalog #200532), 3.0 mM MgCl₂, 200 μM of each dNTP, 5 unitsof a Pfu polymerase deficient in 3′ to 5′ exonuclease activity, taggedor untagged Pfu FEN-1 (˜23 ng), PEF (1 ng) (described in WO 98/42860),PE Biosystems β-Actin primers (300 nM each) (CATALOG #600500), andfluorogenic probe (200 nM; 5′ FAM−3′TAMRA+PE Biosystems catalog #P/N401846) were prepared. 10 ng of human genomic DNA (Promega) was used asthe target nucleic acid sequence in each reaction. Reactions wereperformed in a 50 μl volume. Negative control reactions comprising a Pfupolymerase deficient in both 5′ to 3′ and 3′ to 5′ exonucleaseactivities alone or containing all of the components except the humangenomic DNA template were also prepared. A reaction mixture containing2.5 Units of Taq 2000 was prepared and used as a positive control.Reactions were analyzed in a spectrofluorometric thermocycler (ABI7700). Thermocycling parameters were 95° C. for 2 min and 40 cycles of95° C. for 15 sec, 60° C. for 60 sec and 72° C. for 15 sec.

As demonstrated in FIG. 7, no signal was generated in the presence of aPfu polymerase, naturally deficient in 5′ to 3′ exonuclease activityalone. In the presence of both a Pfu polymerase deficient in 5′ to 3′exonuclease activity and tagged Pfu FEN-1 a signal was generated with athreshold cycle (Ct) of 23 and a final fluorescence intensity (FI) of20,000 units. In the presence of a Pfu polymerase deficient in 5′ to 3′exonuclease activity and untagged Pfu FEN-1 a signal was generated witha Ct of 21 and a final FI of 20,000 units. In the presence of Taq 2000,a signal was generated with a Ct of 21 and a FI of 19,000 units(TaqMan).

These results demonstrate that the presence of β-actin target can bedetected by a PCR assay wherein a signal is generated in the presence ofa Pfu polymerase deficient in 5′ to 3′ exonuclease activity and athermostable FEN-1 nuclease. This signal is comparable to the signalgenerated in the presence of Taq 2000 in the absence of FEN-1. Further,the 5′ to 3′ exonuclease activity that is absent in a Pfu polymerase canbe restored in trans by the addition of Pfu FEN-1.

Example 6

An assay according to the invention involving rolling circleamplification is performed using the human ornithine transcarbamylasegene as a target, which is detected in human DNA extracted from buffycoat by standard procedures. Target (400 ng) is heat-denatured for 4minutes at 97° C., and incubated under ligation conditions in thepresence of two 5′-phosphorylated oligonucleotides, an open circle probeand one gap oligonucleotide. The open circle probe has the sequence:gaggagaataaaagtttctcataagactcgtcatgtctcagcagcttctaacggtcactaatacgactcactataggttctgcctctgggaacac(SEQ ID NO: 17), the gap nucleotide for the wild-type sequence is:tagtgatc. FIGS. 8 and 9 depict rolling circle probes and rolling circleamplification. The reaction buffer (40 ul) contains 5 units/μl of T4 DNAligase (New England Biolabs), 10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 10 mMMgCl₂, 4 mM ATP, 80 nM open circle probe and 100 nM gap oligonucleotide.After incubation for 25 minutes at 37° C., 25 ul are removed and addedto 25 ul of a solution containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1mM DTT, 400 μM each of dTTP, dATP, dGTP, dCTP, 0.2 μM rolling circlereplication primer: gctgagacatgacgagtc (SEQ ID NO: 18), phi29 DNApolymerase (160 ng/50 ul). The sample is incubated for 30 minutes at 30°C.

RNA is produced from a T7 promoter present in the open circle probe, bythe addition of a compensating buffer (a stock solution or concentrate)that is diluted to achieve the following concentration of reagents: 35mM Tris-HCl, pH 8.2, 2 mM spermidine, 18 mm MgCl₂, 5 mM GMP, 1 mM ofATP, CTP, GTP, 333 uM UTP, 667 uM Biotin-16-UTP, 0.03% Tween 20, 2 unitsper ul of T7 RNA polymerase. RNA production is performed as described inU.S. Pat. No. 5,858,033. The incubation is allowed to proceed for 90minutes at 37° C.

Five μl of each sample (the actual test sample, a (−) ligase controlsample, a (−) phi29 DNA polymerase control and a (−)T7 RNA polymerasecontrol) in duplicate are removed for detection. The reversetranscription process includes the steps of A) ligating the open circle,B) synthesizing rolling circle single stranded DNA, C) making RNA (froma T7 promoter present in the open circle probe), D) reverse transcribingthe RNA to make cDNA, and E) performing PCR amplification of the cDNAusing primers and probes for generation of an detection of FEN cleavagestructures, according to the invention. For reverse transcription, thereagents and protocols supplied with the Stratagene Sentinel Single-TubeRT-PCR Core Reagent Kit (Cat# 600505) are used, except for thesubstitution of equal amounts of Yaq DNA polymerase for the Taq 2000 DNApolymerase which is recommended by the manufacturer. Each reactioncontains 1× Sentinel molecular beacon RT-PCR core buffer, 3.5 mM MgCl₂,200 μM of each dNTP, 5 units exo-Pfu, 23 ng Pfu FEN-1, 1 ng PEF, 500 nMeach of the upstream primer: aagtttctcataagactcgtcat (SEQ ID NO: 19),the reverse primer: aggcagaacctatagtgagtcgt (SEQ ID NO: 20), and thefluorogenic probe (for example FAM-DABCYL): agcttctaacggtcactaatacg (SEQID NO: 21). The reactions are subjected to incubation for 30 minutes at45° C., 3 minutes at 95° C., followed by one cycle in a thermal cycler:2 minutes at 95° C., 1 minute at 50° C., 1 minute at 72° C. Thefluorescence in then determined in a fluorescence plate reader, such asStratagene's FluorTracker or PE Biosystems' 7700 Sequence DetectionSystem in Plate-Read Mode.

A crosscheck for the efficiency of detection is possible because of theincorporation of Biotin-16-UTP in the rolling circle amplification RNAproduct. An aliquot of the reactions is captured on glass slides (oralternatively in microwell plates) using an immobilized capture probe.Detection of the captured RNA amplicon is described in detail in U.S.Pat. No. 5,854,033, hereby incorporated by reference.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

1. A method of generating a signal indicative of the presence of atarget nucleic acid in a sample, comprising: in the same reactionvessel, forming a cleavage structure comprising duplex andsingle-stranded nucleic acid by incubating a sample comprising a targetnucleic acid with a thermostable nucleic acid polymerase substantiallylacking 5′ to 3′ exonuclease activity and cleaving said cleavagestructure with a thermostable Fen nuclease lacking 5′ to 3′ syntheticactivity to generate a signal, wherein generation of said signal isindicative of the presence of a target nucleic acid in said sample.
 2. Amethod of detecting and/or measuring a target nucleic acid in a sample,comprising: in the same reaction vessel, forming a cleavage structurecomprising duplex and single-stranded nucleic acid, wherein said singlestranded nucleic acid comprises a 5′ flap, by incubating a samplecomprising a target nucleic acid with a thermostable nucleic acidpolymerase substantially lacking 5′ to 3′ exonuclease activity andcleaving said cleavage structure with a thermostable Fen nucleasesubstantially lacking 5′ to 3′ synthetic activity to release a nucleicacid fragment, anti detecting and/or measuring the release of saidfragment as an indication of the presence of the target in the sample.3. The method of claim 1 or 2, wherein said method comprises, in saidreaction vessel, a pair of amplification primers complementary with saidtarget nucleic acid to permit amplification of a sequence of said targetnucleic acid between said primers.
 4. The method of claim 3, whereinsaid method comprises, in said reaction vessel, one or more dNTPs. 5.The method of claim 3, wherein said method comprises, in said reactionvessel, an oligonucleotide that specifically hybridizes to a region ofthe target nucleic acid between said amplification primers.
 6. Themethod of claims 1 or 2, wherein said method comprises, in said reactionvessel, said thermostable Fen nuclease selected from the groupconsisting of Fen nuclease enzyme of Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus, and Pyrococcushorikoshii.
 7. The method of claims 1 or 2, wherein said methodcomprises, in said reaction vessel, a thermostable polymerase selectedfrom the group consisting of Pfu, Pfu-exo, Taq, Taq-exo, Deep vent andDeep vent exo.
 8. The method of claims 1 or 2, wherein a cleavagestructure is formed prior to the addition of said Fen nuclease, saidcleavage structure comprising a 5′ flap, and said Fen nuclease cleavessaid 5′ flap of said cleavage structure.
 9. The method of claim 8,wherein said 5′ flap comprises a label comprising a first member of apair of interactive labels effectively positioned to quench thegeneration of a detectable signal, the first and second members of saidpair being separable by a site susceptible to Fen nuclease cleavage,thereby allowing the nuclease activity of the Fen nuclease to separatethe first interactive signal generating labeled moiety from the secondinteractive signal generating labeled moiety by cleaving at said sitesusceptible to Fen nuclease, thereby generating a signal.
 10. The methodof claim 9, wherein said pair of interactive labels comprises a quenchermoiety and a fluorescent moiety.
 11. The method of claim 3, wherein saidamplification primers are between about 10 to 100 nucleotides in length.12. The method of claim 3, wherein said amplification primers arebetween about 17-50 nucleotides in length.
 13. The method of claim 3,wherein said amplification primers are between about 17-45 nucleotidesin length.
 14. The method of claim 1, wherein said Fen nuclease is aFen-1 nuclease.